Asymmetric multispecific antibodies

ABSTRACT

The present invention relates to multispecific antibodies, for example bispecific antibodies, and methods for the isolation or purification of the same. The antibodies of the invention comprise first and second heavy chain-light chain pairings wherein each pairing comprises a distinct selective recognition site including one or more amino acid residues contributed from the heavy chain and the light chain of the pairing. The first and second selective recognition sites differ by at least one amino acid residue and can be differentially bound by first and second selective recognition agents according to the methods of the invention. Such methods facilitate the production of antibody preparations enriched for multispecific antibodies having the correct functional heavy chain-light chain pairings.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/503,602, filed Aug. 18, 2017, now U.S. Pat. No. 10,487,156, which isa 35 U.S.C. § 371 filing of International Patent Application No.PCT/EP2015/069186, filed Aug. 20, 2015, which claims priority to UnitedKingdom Patent Application No. 1414823.3, filed Aug. 20, 2014, theentire disclosures of which are hereby incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to multispecific antibodies, for examplebispecific antibodies, and methods for the isolation or purification ofthe same. The antibodies and methods of the invention facilitate theproduction of antibody preparations enriched for multispecificantibodies having the correct functional heavy chain-light chainpairings required for the antibody to bind two or more epitopes on thesame antigen or different antigens.

BACKGROUND TO THE INVENTION

Naturally occurring antibodies, including bivalent antibodies, exhibitimmunoreactivity to a specific epitope on a particular target antigen.Multispecific antibodies, as the name indicates, are antibodiesengineered to recognise and bind more than one epitope, potentially ondifferent target antigens of interest.

Naturally occurring antibodies typically include combinations of heavyand light immunoglobulin chains, wherein the antigen binding propertiesof the molecule are determined by the variable regions or domains of theheavy and light chains i.e. the VH and VL domains, respectively. Morespecifically, the antigen binding sites of any antibody typicallyinclude residues contributed by three complementarity determiningregions (CDRs) within each of the VH and VL domains.

Multispecific antibodies differ from naturally occurring antibodies inthat they incorporate more than one VH-VL domain pairing such that theycan recognise and bind to more than one epitope. Commercially theseantibodies are extremely important for their ability to bind more thanone target antigen. However, significant difficulties exist in themanufacture and isolation of multispecific antibodies as a result ofmispairings between the different heavy chains and light chainsincorporated into the same antibody molecule. These mispairings can leadto the inadvertent production of monospecific antibodies or antibodieshaving non-functional or non-productive antigen binding sites, therebyreducing the yield of the multispecific antibody of interest.

FIG. 1 illustrates the difficulties that can arise in the production ofa bispecific antibody exhibiting immunoreactivity for two distinctepitopes. The bispecific antibody as shown (A) includes two distinctheavy chains and two distinct light chains. However, only the correctpairing of these four immunoglobulin chains gives rise to an antibodyhaving the required binding profile i.e. specificity for both targetantigens. There are in fact nine other potential combinations that canform from a mixture of the four heavy and light chains shown, whichresult in bivalent monospecific antibodies (E and H), monovalentmonospecific antibodies (B, C, G and J) and non-binding antibodies (D, Fand I). This problem becomes worse the more complex the multispecificantibody molecule i.e. the more epitopes or antigens the antibody isintended to bind.

Various attempts have been made to improve multispecific antibodyproduction by addressing the problem of incorrect chain pairing. Severalapproaches have focussed on engineering antibodies so as to promote thecorrect pairing between VH-VL domains. For instance, US2010/0254989A1describes the construction of bispecific cMet-ErbB1 antibodies, wherethe VH and VL of the individual antibodies are fused genetically via aGlySer linker. An alternative approach uses rat-mouse quadromas forgenerating bispecific antibodies, where the mouse and the rat antibodypredominantly forms the original VH-VL pairings and the bispecificantibody consists of the rat and the mouse Fc (Lindhofer et al., JImmunol. (1995) 155: 1246-1252).

For bispecific antibodies including an Fc domain, researchers have alsofocussed on introducing mutations into the constant region of the heavychains to promote the correct heterodimerization of the Fc portion.Several such techniques are reviewed in Klein et al. (mAbs (2012) 4:6,1-11), the contents of which are incorporated herein by reference intheir entirety. These techniques include the “knobs-into-holes” (KiH)approach which involves the introduction of a bulky residue into one ofthe CH3 domains of one of the antibody heavy chains. This bulky residuefits into a complementary “hole” in the other CH3 domain of the pairedheavy chain so as to promote correct pairing of heavy chains.

Researchers have also attempted to resolve the problem of achievingcorrect association of heavy chain and light chain pairs. One approachuses the CrossMab principle (as reviewed in Klein et al.), whichinvolves domain swapping between heavy and light chains so as to promotethe formation of the correct pairings. Others have sought to engineerthe interfaces between the paired VH-VL domains or paired CH1-CL domainsof the heavy and light chains so as to increase the affinity between theheavy chain and its cognate light chain (Lewis et al. NatureBiotechnology (2014) 32: 191-198). Techniques such as those describedabove that require extensive antibody engineering have met with somesuccess; however, the production of antibodies harbouring specificmutations can be labour intensive and can result in antibodies which arehighly immunogenic in humans and/or suffer from a loss of effectorfunction.

An alternative approach to the production of multispecific antibodypreparations having the correct antigen specificity has been thedevelopment of methods that enrich for antibodies having the correctheavy chain-light chain pairings. For example, Spiess et al. (NatureBiotechnology (2013) 31: 753-758) describe a method for the productionof a MET-EGFR bispecific antibody from a co-culture of bacteriaexpressing two distinct half-antibodies. Methods have also beendescribed wherein the constant region of at least one of the heavychains of a bispecific antibody is mutated so as to alter its bindingaffinity for an affinity agent, for example Protein A. This allowscorrectly paired heavy chain heterodimers to be isolated based on apurification technique that exploits the differential binding of the twoheavy chains to an affinity agent (see US2010/0331527, WO2013/136186).The limitation with methods that select for correct heavy chainheterodimerization based on differential binding is that they do notselect for antibodies having the correct heavy chain-light chainpairings such that these techniques are typically applied tomultispecific antibodies having a shared or common light chain.

International patent application no. PCT/EP2012/071866 (WO2013/064701)addresses the problem of incorrect chain pairing using a method formultispecific antibody isolation based on the use of anti-idiotypicbinding agents, in particular anti-idiotypic antibodies. Theanti-idiotypic binding agents are employed in a two-step selectionmethod in which a first agent is used to capture antibodies having aVH-VL domain pairing specific for a first antigen and a second agent issubsequently used to capture antibodies also having a second VH-VLdomain pairing specific for a second antigen.

The drawback with this method is that the anti-idiotypic binding agentsused to isolate the antibody must be specific for each multispecificantibody produced, depending on its antigen binding profile. Therefore,although the principle of the method described in PCT/EP2012/071866 isgenerally applicable to the isolation of any multispecific antibody, thereagents i.e. the anti-idiotypic binding agents, must be generated inaccordance with the specific VH-VL domain pairings of the multispecificantibody to be isolated.

SUMMARY OF INVENTION

The present invention improves upon the state of the art by providingmultispecific antibodies having at least two distinct heavy chain-lightchain pairings wherein the pairings can be distinguished by the presenceof selective recognition sites. Each selective recognition sitecomprises residues contributed by both the heavy and light chain of thepairing, but does not include residues located within the antigenbinding site.

In a first aspect, the present invention provides a multispecificantibody comprising

-   -   (i) a first pairing comprising a first heavy chain paired with a        first light chain, wherein the first pairing comprises a first        antigen binding site; and    -   (ii) a second pairing comprising a second heavy chain paired        with a second light chain wherein the second pairing comprises a        second antigen binding site,        characterised in that the first pairing comprises a first        selective recognition site comprising at least one amino acid        residue from the first heavy chain and at least one amino acid        residue from the first light chain and the second pairing        comprises a second selective recognition site comprising at        least one amino acid residue from the second heavy chain and at        least one amino acid residue from the second light chain,        wherein the first and second selective recognition sites do not        include residues from the antigen binding site of the first or        second pairing,        and wherein the combination of amino acid residues of the first        selective recognition site differs by at least one amino acid        residue from the combination of amino acid residues of the        second selective recognition site such that the first and second        selective recognition sites can be differentially bound by first        and second selective recognition agents.

The present invention also provides multispecific antibodies having atleast two distinct heavy chain-light chain pairings wherein the firstand second heavy chains differ by at least one amino acid residue in theconstant region, preferably in the CH1 domain. The first and secondheavy chains can differ because each chain comprises a fragment derivedfrom the constant region of a different human immunoglobulin subtype ora different allotype, preferably different human allotypes. In certainembodiments, the first and second heavy chains comprise the entireconstant region from different human immunoglobulin subtypes ordifferent allotypes, wherein the constant regions differ by at least oneamino acid residue in the CH1 domain. These differences can be exploitedsuch that multispecific antibodies having the correct heavy chain-lightchain pairings can be identified and purified.

Thus, provided herein is a multispecific antibody comprising

-   -   (i) a first pairing comprising a first heavy chain paired with a        first light chain, wherein the first pairing comprises a first        antigen binding site; and    -   (ii) a second pairing comprising a second heavy chain paired        with a second light chain wherein the second pairing comprises a        second antigen binding site,        characterised in that the first and second heavy chain each        comprise a fragment derived from the constant region of a        different human immunoglobulin subtype, wherein the fragments        differ by at least one amino acid residue.

Further provided is a multispecific antibody comprising

-   -   (i) a first pairing comprising a first heavy chain paired with a        first light chain, wherein the first pairing comprises a first        antigen binding site; and    -   (ii) a second pairing comprising a second heavy chain paired        with a second light chain wherein the second pairing comprises a        second antigen binding site,        characterised in that the first and second heavy chain each        comprise a fragment derived from the constant region of a        different immunoglobulin allotype, wherein the fragments differ        by at least one amino acid residue.

Preferably, the fragments derived from the constant region of thedifferent human immunoglobulin subtypes or the different allotypescomprise or consist of the CH1 domain.

Multispecific antibodies of the present invention can be isolatedaccording to the methods provided herein. These methods exploit thevariation that can be found in immunoglobulin heavy and light chainsoutside of the antigen binding site, and in particular, the naturalvariation found in the constant regions of heavy chain-light chainpairings, so as to produce multispecific antibody preparations enrichedfor antibodies having the correct functional VH-VL pairings required fortarget antigen recognition and binding.

The methods of the present invention achieve isolation and/orpurification of multispecific antibodies using selective recognitionagents that recognise the selective recognition sites within thedistinct immunoglobulin heavy chain-light chain pairings. The fact thatthe methods of the invention are based on the selective binding ofagents to regions of the antibody outside the antigen binding sites,means that the methods are broadly applicable to the purification ofmultispecific antibodies irrespective of the epitopes or antigens towhich the antibody is designed to bind.

In a further aspect, the present invention provides a method for theisolation of a multispecific antibody according to the first aspect ofthe invention from a sample, said method comprising:

-   -   a. contacting the sample with a first selective recognition        agent which selectively binds to the first selective recognition        site;    -   b. releasing any antibodies bound to the first selective        recognition agent to obtain a second sample containing        antibodies having the first heavy chain-light chain pairing;    -   c. contacting the second sample with a second selective        recognition agent which selectively binds to the second        selective recognition site;    -   d. releasing any antibodies bound to the second selective        recognition agent thereby obtaining a preparation of antibodies        having both first and second heavy chain-light chain pairings.

The invention also relates to reagents and kits for producingmultispecific antibodies according to the invention, and also reagents,particularly antibodies, and kits for isolating multispecific antibodiesusing the methods of the invention.

In a further aspect, the present invention provides an antibody orantigen binding fragment thereof that binds to a multispecific antibodyaccording to any of the preceding aspects of the invention, wherein theantibody or antigen binding fragment thereof binds to an epitope,wherein the epitope is:

-   -   (i) comprised within the first pairing (the first heavy chain        paired with the first light chain) but not the second pairing        (the second heavy chain paired with the second light chain); or    -   (ii) comprised within the second pairing (the second heavy chain        paired with the second light chain) but not the first pairing        (the first heavy chain paired with the first light chain) and        wherein the epitope does not include residues from the antigen        binding site.

In a further aspect, the present invention provides a kit for producinga multispecific antibody according to the first aspect of the inventioncomprising one or more polynucleotides, wherein the polynucleotide(s)encode(s) fragments of the first and second heavy chains and fragmentsof the first and second light chains wherein the fragments contain atleast the residues of the first and second selective recognition sites.Also provided herein is a kit for isolating a multispecific antibodyaccording to the method aspect of the invention wherein the kitcomprises a first selective recognition agent and a second selectiverecognition agent. Further provided is a kit for producing and isolatinga multispecific antibody of the present invention wherein the kitcomprises:

-   -   (i) one or more polynucleotides, wherein the polynucleotide(s)        encode(s) fragments of the first and second heavy chains and        fragments of the first and second light chains wherein the        fragments contain at least the residues of the first and second        selective recognition sites; and    -   (ii) a first selective recognition agent and a second selective        recognition agent wherein the first and second selective        recognition agents can selectively bind the first and second        selective recognition sites within the fragments encoded by the        one or more polynucleotides of (i).

These, and other, embodiments of the invention will be betterappreciated and understood when considered in conjunction with thefollowing description and the accompanying drawings. It should beunderstood, however, that the following description, while indicatingvarious embodiments of the invention and numerous specific detailsthereof, is given by way of illustration and not of limitation. Manysubstitutions, modifications, additions and/or rearrangements may bemade within the scope of the invention without departing from the spiritthereof, and the invention includes all such substitutions,modifications, additions and/or rearrangements.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the ten different pairings of heavy and light chaincombinations that can result from the production of a bispecificantibody having two distinct heavy chains and two distinct light chains.

FIG. 2 Immunoglobulin structure showing heavy and light chainpolypeptides and the variable (VL and VH) and constant domains (CL andCH1, CH2 and CH3) of each.

FIG. 3 Alignment of CH1 domains derived from human IgG heavy chainsubtypes and allotypes demonstrating sequence variation betweendifferent allotypes. Sequences, from top to bottom, correspond to SEQ IDNOs: 1-9, respectively.

FIG. 4 Crystal structure of human IgG heavy chain CH1 domains pairedwith human light chain CL domains. (A) CH1 domain of an IgG1 moleculepaired with the CL domain of a lambda light chain; (B) CH1 domain of anIgG2 molecule paired with the CL domain of a kappa light chain.

FIG. 5 Alignment of CL domains derived from human lambda chain allotypesdemonstrating sequence variation between different allotypes. Sequences,from top to bottom, correspond to SEQ ID NOs: 10, 11, 12, 14, and 13,respectively.

FIG. 6 Crystal structure of the CL domain of the human lambda lightchain paired with the CH1 domain of human IgG1.

FIG. 7 Exemplary bispecific antibodies of the invention having CH1domains that differ by at least one amino acid residue and CL domainsthat differ by virtue of the fact that they are derived from the lambdaand kappa light chains.

FIG. 8 (A) An exemplary composite bispecific IgG antibody of theinvention incorporating fragments derived from the CH1 domains ofdifferent human immunoglobulin heavy chain allotypes. The fragmentscomprise residues 137 and 138 of the heavy chain allotypes, wherein theamino acids at positions 137 and 138 differ between the allotypes. (B)An exemplary bispecific IgG antibody of the invention incorporatingamino acid substitutions derived from naturally occurring humanimmunoglobulin allotypes in the CH1 domains of the heavy chains.

FIG. 9 Schematic of a method for isolating bispecific antibodiesaccording to the invention. In step 1, a mixture of bispecificantibodies having various heavy chain-light chain pairings is contactedwith a first selective recognition agent that selectively binds to afirst selective recognition site comprised within the first heavychain-light chain pairing of interest, but outside the antigen bindingsite. Antibodies bound to the first selective recognition agent arereleased to produce a second sample. In step 2, the second sample iscontacted with a second selective recognition agent that selectivelybinds to a second selective recognition site comprised within the secondheavy chain-light chain pairing of interest. Antibodies bound to thesecond selective recognition agent can be released to yield bispecificantibodies having the correct heavy chain-light chain pairings andtherefore the correct antigen binding profile.

FIG. 10 Phage display selection for VHH antibodies with bindingspecificity for antibodies having mutations in the CH1 domain. Resultsof phage titration on E. coli TG1 cells following selection ofVHH-expressing phage using the method described in Example 3 andsummarised in Table 12.

FIG. 11 Phage display selection for VHH antibodies with bindingspecificity for antibodies having mutations in the CH1 domain. Resultsof phage titration on E. coli TG1 cells following selection ofVHH-expressing phage using the method described in Example 5 andsummarised in Table 15.

DETAILED DESCRIPTION A. Definitions

“Antibody” and “Immunoglobulin”

As used herein, the term “immunoglobulin” includes a molecule having acombination of two heavy chains and two light chains. An “antibody” or“antibody molecule” refers to an immunoglobulin with a specificimmunoreactive activity to an antigen of interest (e.g. a humanantigen). Antibodies and immunoglobulins comprise light and heavychains, with or without an interchain covalent linkage between them.Basic immunoglobulin structures in vertebrate systems are relativelywell understood.

The generic term “immunoglobulin” comprises five distinct classes ofantibody that can be distinguished biochemically. All five classes ofantibodies are within the scope of the present invention, although thefollowing discussion will generally be directed to the IgG class ofimmunoglobulin molecules. Naturally occurring IgG immunoglobulinscomprise two identical light polypeptide chains of molecular weightapproximately 23,000 Daltons, and two identical heavy polypeptide chainsof molecular weight 53,000-70,000. The four chains are joined bydisulfide bonds in a “Y” configuration wherein the light chains bracketthe heavy chains starting at the mouth of the “Y” and continuing throughthe variable region (see FIG. 2 ).

The light chains of an antibody are classified as either kappa or lambda(κ, λ). Each heavy chain class may be bound with either a kappa orlambda light chain. In general, the light and heavy chains arecovalently bonded to each other, and the “tail” portions of the twoheavy chains are bonded to each other by covalent disulfide linkages ornon-covalent linkages when the immunoglobulins are generated either byhybridomas, B cells or genetically engineered host cells.

In the heavy chain, the amino acid sequence runs from an N-terminus atthe forked end of the Y configuration to the C-terminus at the bottom ofeach chain. Those skilled in the art will appreciate that heavy chainsare classified as gamma, mu, alpha, delta, or epsilon, (γ, μ, α, δ, ε)with some subclasses or subtypes among them (e.g., γ1-γ4). It is thenature of this chain that determines the “class” of the antibody as IgG,IgM, IgA, IgD or IgE, respectively. The immunoglobulin subclasses(subtypes or isotypes) e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc. are wellcharacterized and are known to confer functional specialization.Modified versions of each of these classes and isotypes are readilydiscernable to the skilled artisan in view of the instant disclosureand, accordingly, are within the scope of the instant invention.

“Variable Region” and “Variable Domains”

The variable region of an antibody is the portion which allows theantibody to selectively recognise and specifically bind epitopes onantigens. The variable region is typically made up of the variabledomain of the heavy chain (VH) and the variable domain of the lightchain (VL), both domains of which are located at the N terminus of theirrespective polypeptides (see FIG. 2 ).

The term “variable” refers to the fact that certain portions of thevariable domains VH and VL differ extensively in sequence amongantibodies and are used in the binding and specificity of eachparticular antibody for its target antigen. However, the variability isnot evenly distributed throughout the variable domains of antibodies. Itis concentrated in three segments called “hypervariable loops” in eachof the VL domain and the VH domain which form part of the antigenbinding site. The first, second and third hypervariable loops of theVLambda light chain domain may be defined as comprising residues 24-33(L1(λ), consisting of 9, 10 or 11 amino acid residues), 49-53 (L2(λ),consisting of 3 residues) and 90-96 (L3(λ), consisting of 5 residues) inthe VL domain (Morea et al., Methods 20:267-279 (2000)). The first,second and third hypervariable loops of the VKappa light chain domainmay be defined as comprising residues 25-33 (L1(κ), consisting of 6, 7,8, 11, 12 or 13 residues), 49-53 (L2(κ), consisting of 3 residues) and90-97 (L3(κ), consisting of 6 residues) in the VL domain (Morea et al.,Methods 20:267-279 (2000)). The first, second and third hypervariableloops of the VH domain may be defined as comprising residues 25-33 (H1,consisting of 7, 8 or 9 residues), 52-56 (H2, consisting of 3 or 4residues) and 91-105 (H3, highly variable in length) in the VH domain(Morea et al., Methods 20:267-279 (2000)).

The hypervariable loops (L1, L2, L3, H1, H2 and H3) may each comprisepart of a “complementarity determining region” or “CDR”, as definedbelow. The terms “hypervariable loop” and “complementarity determiningregion” are not strictly synonymous, since the hypervariable loops (HVs)are defined on the basis of structure, whereas complementaritydetermining regions (CDRs) are defined based on sequence variability(Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.,1983).

The CDRs of the VL and VH domains can typically be defined as comprisingthe following amino acids: residues 24-34 (LCDR1), 50-56 (LCDR2) and89-97 (LCDR3) in the light chain variable domain, and residues 31-35 or31-35b (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3) in the heavy chainvariable domain; (Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)). Thus, the HVs may be comprised within thecorresponding CDRs and references herein to the “hypervariable loops” ofVH and VL domains should be interpreted as also encompassing thecorresponding CDRs, and vice versa, unless otherwise indicated.

The more highly conserved portions of variable domains are called theframework region (FR), as defined below. The variable domains of nativeheavy and light chains each comprise four FRs (FR1, FR2, FR3 and FR4,respectively), largely adopting a β-sheet configuration, connected bythe three hypervariable loops. The hypervariable loops in each chain areheld together in close proximity by the FRs and, with the hypervariableloops from the other chain, contribute to the formation of the antigenbinding site of antibodies. Structural analysis of antibodies revealedthe relationship between the sequence and the shape of the binding siteformed by the complementarity determining regions (Chothia et al., J.Mol. Biol. 227: 799-817 (1992)); Tramontano et al., J. Mol. Biol,215:175-182 (1990)). Despite their high sequence variability, five ofthe six loops adopt just a small repertoire of main-chain conformations,called “canonical structures”. These conformations are first of alldetermined by the length of the loops and secondly by the presence ofkey residues at certain positions in the loops and in the frameworkregions that determine the conformation through their packing, hydrogenbonding or the ability to assume unusual main-chain conformations.

“CDR”

As used herein, the term “CDR” or “complementarity determining region”means the non-contiguous antigen binding sites found within the variableregion of both heavy and light chain polypeptides. These particularregions have been described by Kabat et al., J. Biol. Chem. 252,6609-6616 (1977) and Kabat et al., Sequences of protein of immunologicalinterest. (1991), and by Chothia et al., J. Mol. Biol. 196:901-917(1987) and by MacCallum et al., J. Mol. Biol. 262:732-745 (1996) wherethe definitions include overlapping or subsets of amino acid residueswhen compared against each other. The amino acid residues whichencompass the CDRs as defined by each of the above cited references areset forth for comparison. Preferably, the term “CDR” is a CDR as definedby Kabat based on sequence comparisons.

TABLE 1 CDR definitions CDR Definitions Kabat¹ Chothia² MacCallum³ V_(H)CDR1 31-35 26-32 30-35 V_(H) CDR2 50-65 53-55 47-58 V_(H) CDR3  95-102 96-101  93-101 V_(L) CDR1 24-34 26-32 30-36 V_(L) CDR2 50-56 50-5246-55 V_(L) CDR3 89-97 91-96 89-96 ¹Residue numbering follows thenomenclature of Kabat et al., supra ²Residue numbering follows thenomenclature of Chothia et al., supra ³Residue numbering follows thenomenclature of MacCallum et al., supra

“Framework Region”

The term “framework region” or “FR region” as used herein, includes theamino acid residues that are part of the variable region, but are notpart of the CDRs (e.g., using the Kabat definition of CDRs). Therefore,a variable region framework is between about 100-120 amino acids inlength but includes only those amino acids outside of the CDRs. For thespecific example of a heavy chain variable domain and for the CDRs asdefined by Kabat et al., framework region 1 corresponds to the domain ofthe variable region encompassing amino acids 1-30; framework region 2corresponds to the domain of the variable region encompassing aminoacids 36-49; framework region 3 corresponds to the domain of thevariable region encompassing amino acids 66-94, and framework region 4corresponds to the domain of the variable region from amino acids 103 tothe end of the variable region. The framework regions for the lightchain are similarly separated by each of the light claim variable regionCDRs. Similarly, using the definition of CDRs by Chothia et al. orMcCallum et al. the framework region boundaries are separated by therespective CDR termini as described above. In preferred embodiments theCDRs are as defined by Kabat.

The framework regions largely adopt a β-sheet conformation and the CDRsform loops which connect, and in some cases form part of, the β-sheetstructure. Thus, these framework regions act to form a scaffold thatprovides for positioning the six CDRs in correct orientation byinter-chain, non-covalent interactions.

“Antigen Binding Site”

As used herein, the “antigen binding site” is the site within thevariable region of the antibody which allows the antibody to selectivelyrecognise and specifically bind epitopes on antigens. In naturallyoccurring antibodies, the six CDRs present on each monomeric antibodyare short, non-contiguous sequences of amino acids that are specificallypositioned to form the antigen binding site as the antibody assumes itsthree dimensional configuration in an aqueous environment. As usedherein, “residues from the antigen binding site” correspond to residueslocated within one or more of the CDRs present in either the VH or VLdomain. The antigen binding site formed by the positioned CDRs defines asurface complementary to the epitope on the immunoreactive antigen. Thiscomplementary surface promotes the non-covalent binding of the antibodyto the immunoreactive antigen epitope. The position of CDRs can bereadily identified by one of ordinary skill in the art.

“Epitope”

The term “epitope” refers to a specific arrangement of amino acidslocated on a peptide or protein to which an antibody or antibodyfragment binds. Epitopes often consist of a chemically active surfacegrouping of molecules such as amino acids or sugar side chains, and havespecific three dimensional structural characteristics as well asspecific charge characteristics. Epitopes can be linear, i.e., involvingbinding to a single sequence of amino acids, or conformational, i.e.,involving binding to two or more sequences of amino acids in variousregions of the antigen that may not necessarily be contiguous.

“Constant Region” and “Constant Domains”

The constant region of an antibody includes the portion of each of theheavy and light chain polypeptides outside of the variable region.Immunoglobulin light chains typically include a single constant domain(denoted herein as CL or CL1) which lies C terminal to the VL domain.Immunoglobulin heavy chains typically include a constant regionconsisting of a first constant domain (CH1) a hinge region, a secondconstant domain (CH2) and a third constant domain (CH3). As shown inFIG. 2 , these domains are positioned C terminal to the VH domain.

The numbering of the amino acids in the heavy and light immunoglobulinchains run from the N-terminus at the forked ends of the Y configurationto the C-terminus at the bottom of each chain. Unless otherwisespecified, specific amino acid positions of the heavy chain constantdomains are identified herein with reference to the EU numbering schemefor human IgG1 molecules (see Tables 2-4 below). For the kappa andlambda light chains, specific amino acid positions within the constantregion are defined with reference to the nomenclature of Kabat et al.,supra used for human light chains (see Tables 5 and 6 below)

The Tables below show the correspondence between the different numberingschemes used to identify the residues in the CH1, CH2 and CH3 domains ofthe human IgG1 heavy chain and the CL domains of the human kappa andhuman lambda light chains. It would be within the capabilities of theskilled person to align the sequence of a heavy chain or light chainpolypeptide of different origin with the sequences shown in the Tablesbelow in order to identify corresponding or equivalent residues.

TABLE 2 Amino acid residues of human IgG1 CH1 domain CH1 IMGT EUStrands, unique IMGT exon number- turns and number- IGHG1 number- ingKabat loops for ing for amino acid ing (98 aa) number- C-domainC-domains translation¹ 1-98 118-215 ing 1.8 — — — — 1.7 — — — — 1.6 — —— — 1.5 — — — — 1.4 (A)  1 118 114 1.3 S  2 119 115 1.2 T  3 120 116 1.1K  4 121 117 A-STRAND 1 G  5 122 118 2 P  6 123 119 3 S  7 124 120 4 V 8 125 121 5 F  9 126 122 6 P 10 127 123 7 L 11 128 124 8 A 12 129 125 9P 13 130 126 10 S 14 131 127 11 S 15 132 128 12 K 16 133 129 13 S 17 134130 14 T 18 135 133 15 S 19 136 134 AB-TURN 15.1 — — — — 15.2 — — — —15.3 — — — — B-STRAND 16 G 20 137 135 17 G 21 138 136 18 T 22 139 137 19A 23 140 138 20 A 24 141 139 21 L 25 142 140 22 G 26 143 141 23 C 27 144142 24 L 28 145 143 25 V 29 146 144 26 K 30 147 145 BC-LOOP 27 D 31 148146 28 Y 32 149 147 29 F 33 150 148 30 P 34 151 149 31 — — — — 34 — — —— 35 E 35 152 150 36 P 36 153 151 37 V 37 154 152 38 T 38 155 153C-STRAND 39 V 39 156 154 40 S 40 157 156 41 W 41 158 157 42 N 42 159 16243 S 43 160 163 44 G 44 161 164 45 A 45 162 165 CD- 45.1 L 46 163 166STRAND 45.2 T 47 164 167 45.3 S 48 165 168 45.4 — — — — 45.5 — — — —45.6 — — — — 45.7 — — — — D-STRAND 77 G 49 166 169 78 V 50 167 171 79 H51 168 172 80 T 52 169 173 81 F 53 170 174 82 P 54 171 175 83 A 55 172176 84 V 56 173 177 DE-TURN 84.1 L 57 174 178 84.2 Q 58 175 179 84.3 S59 176 180 84.4 S 60 177 182 84.5 — — — — 84.6 — — — — 84.7 — — — — 85.7— — — — 85.6 — — — — 85.5 — — — — 85.4 G 61 178 183 85.3 L 62 179 18485.2 Y 63 180 185 85.1 S 64 181 186 E-STRAND 85 L 65 182 187 86 S 66 183188 87 S 67 184 189 88 V 68 185 190 89 V 69 186 191 90 T 70 187 192 91 V71 188 193 92 P 72 189 194 93 S 73 190 195 94 S 74 191 196 95 S 75 192197 96 L 76 193 198 EF-TURN 96.1 — — — — 96.2 — — — — F-STRAND 97 — — —— 98 G 77 194 199 99 T 78 195 200 100 Q 79 196 203 101 T 80 197 205 102Y 81 198 206 103 I 82 199 207 104 C 83 200 208 FG-LOOP 105 N 84 201 209106 V 85 202 210 107 N 86 203 211 108 H 87 204 212 109 K 88 205 213 110P 89 206 214 111 — — — — 112 — — — — 113 S 90 207 215 114 N 91 208 216115 T 92 209 217 116 K 93 210 218 117 V 94 211 219 G-STRAND 118 D 95 212220 119 K 96 213 221 120 K 97 214 222 121 V 98 215 223 122 — — — — 123 —— — — 124 — — — — 125 — — — — 126 — — — — 127 — — — — 128 — — — — 129 —— — — 130 — — — — ¹http://www.imgt.org/ligmdb/view?id=J00228

TABLE 3 Amino acid residues of human IgG1 CH2 domain CH2 IMGT EUStrands, unique IMGT exon number- turns and number- IGHG1 number- ingKabat loops for ing for amino acid ing (110 aa) number- C-domainsC-domains translation¹ 1-110 231-340 ing 1.8 — — — — 1.7 — — — — 1.6 (A)1 231 244 1.5 P 2 232 245 1.4 E 3 233 246 1.3 L 4 234 247 1.2 L 5 235248 1.1 G 6 236 249 A-STRAND 1 G 7 237 250 2 P 8 238 251 3 S 9 239 252 4V 10 240 253 5 F 11 241 254 6 L 12 242 255 7 F 13 243 256 8 P 14 244 2579 P 15 245 258 10 K 16 246 259 11 P 17 247 260 12 K 18 248 261 13 D 19249 262 14 T 20 250 263 15 L 21 251 264 AB-TURN 15.1 M 22 252 265 15.2 I23 253 266 15.3 — — — — B-STRAND 16 S 24 254 267 17 R 25 255 268 18 T 26256 269 19 P 27 257 270 20 E 28 258 271 21 V 29 259 272 22 T 30 260 27323 C 31 261 274 24 V 32 262 275 25 V 33 263 276 26 V 34 264 277 BC-LOOP27 D 35 265 278 28 V 36 266 279 29 S 37 267 280 30 H 38 268 281 31 E 39269 282 34 D 40 270 283 35 P 41 271 284 36 E 42 272 285 37 V 43 273 28638 K 44 274 287 C-STRAND 39 F 45 275 288 40 N 46 276 289 41 W 47 277 29042 Y 48 278 291 43 V 49 279 292 44 D 50 280 295 45 G 51 281 296 CD- 45.1V 52 282 299 STRAND 45.2 E 53 283 300 45.3 V 54 284 301 45.4 H 55 285302 45.5 — — — — 45.6 — — — — 45.7 — — — — D-STRAND 77 N 56 286 303 78 A57 287 304 79 K 58 288 305 80 T 59 289 306 81 K 60 290 307 82 P 61 291308 83 R 62 292 309 84 E 63 293 310 DE-TURN 84.1 E 64 294 311 84.2 Q 65295 312 84.3 Y 66 296 313 84.4 N 67 297 314 84.5 — — — — 84.6 — — — —84.7 — — — — 85.7 — — — — 85.6 — — — — 85.5 — — — — 85.4 S 68 298 31785.3 T 69 299 318 85.2 Y 70 300 319 85.1 R 71 301 320 E-STRAND 85 V 72302 321 86 V 73 303 322 87 S 74 304 323 88 V 75 305 324 89 L 76 306 32590 T 77 307 326 91 V 78 308 327 92 L 79 309 328 93 H 80 310 329 94 Q 81311 330 95 D 82 312 331 96 W 83 313 332 EF-TURN 96.1 — — — — 96.2 — — —— F-STRAND 97 L 84 314 333 98 N 85 315 334 99 G 86 316 335 100 K 87 317336 101 E 88 318 337 102 Y 89 319 338 103 K 90 320 339 104 C 91 321 340FG-LOOP 105 K 92 322 341 106 V 93 323 342 107 S 94 324 343 108 N 95 325344 109 K 96 326 345 110 A 97 327 346 111 — — — — 112 — — — — 113 L 98328 347 114 P 99 329 348 115 A 100 330 349 116 P 101 331 350 117 I 102332 351 G-STRAND 118 E 103 333 352 119 K 104 334 353 120 T 105 335 354121 I 106 336 355 122 S 107 337 357 123 K 108 338 358 124 A 109 339 359125 K 110 340 360 126 — — — — 127 — — — — 128 — — — — 129 — — — — 130 —— — — ¹http://www.imgt.org/ligmdb/view?id=J00228

TABLE 4 Amino acid residues of human IgG1 CH3 domain CH3 IMGT EUStrands, unique IMGT exon number- turns and number- IGHG1 number- ingKabat loops for ing for amino acid ing (107 aa) number- C-domainsC-domains translation¹ 1-107 341-446 ing 1.8 — — — — 1.7 — — — — 1.6 — —— — 1.5 — — — — 1.4 (G) 1 341 361 1.3 Q 2 342 363 1.2 P 3 343 364 1.1 R4 344 365 A-STRAND 1 E 5 345 366 2 P 6 346 367 3 Q 7 347 368 4 V 8 348369 5 Y 9 349 370 6 T 10 350 371 7 L 11 351 372 8 P 12 352 373 9 P 13353 374 10 S 14 354 375 11 R 15 355 376 12 D 16 356 377 13 E 17 357 37814 L 18 358 381 15 T 19 359 382 AB-TURN 15.1 — — — — 15.2 — — — — 15.3 —— — — B-STRAND 16 K 20 360 383 17 N 21 361 384 18 Q 22 362 385 19 V 23363 386 20 S 24 364 387 21 L 25 365 388 22 T 26 366 389 23 C 27 367 39024 L 28 368 391 25 V 29 369 392 26 K 30 370 393 BC-LOOP 27 G 31 371 39428 F 32 372 395 29 Y 33 373 396 30 P 34 374 397 31 — — — — 34 — — — — 35S 35 375 398 36 D 36 376 399 37 I 37 377 400 38 A 38 378 401 C-STRAND 39V 39 379 402 40 E 40 380 405 41 W 41 381 406 42 E 42 382 407 43 S 43 383408 44 N 44 384 410 45 G 45 385 411 CD- 45.1 Q 46 386 414 STRAND 45.2 P47 387 415 45.3 E 48 388 416 45.4 N 49 389 417 45.5 — — — — 45.6 — — — —45.7 — — — — D-STRAND 77 N 50 390 418 78 Y 51 391 419 79 K 52 392 420 80T 53 393 421 81 T 54 394 422 82 P 55 395 423 83 P 56 396 424 84 V 57 397425 DE-TURN 84.1 L 58 398 426 84.2 D 59 399 427 84.3 S 60 400 428 84.4 D61 401 430 84.5 — — — — 84.6 — — — — 84.7 — — — — 85.7 — — — — 85.6 — —— — 85.5 — — — — 85.4 G 62 402 433 85.3 S 63 403 434 85.2 F 64 404 43585.1 F 65 405 436 E-STRAND 85 L 66 406 437 86 Y 67 407 438 87 S 68 408439 88 K 69 409 440 89 L 70 410 441 90 T 71 411 442 91 V 72 412 443 92 D73 413 444 93 K 74 414 445 94 S 75 415 446 95 R 76 416 447 96 W 77 417448 EF-TURN 96.1 — — — — 96.2 — — — — F-STRAND 97 Q 78 418 449 98 Q 79419 450 99 G 80 420 451 100 N 81 421 452 101 V 82 422 453 102 F 83 423454 103 S 84 424 455 104 C 85 425 456 FG-LOOP 105 S 86 426 457 106 V 87427 458 107 M 88 428 459 108 H 89 429 460 109 E 90 430 461 110 A 91 431462 111 — — — — 112 L 92 432 463 113 H 93 433 464 114 N 94 434 465 115 H95 435 466 116 Y 96 436 467 117 T 97 437 468 G-STRAND 118 Q 98 438 469119 K 99 439 470 120 S 100 440 471 121 L 101 441 472 122 S 102 442 473123 L 103 443 474 124 S 104 444 475 125 P 105 445 476 126 — — — — 127 —— — — 128 — — — — 129 G CHS 106 446 477 130 K CHS 107 447¹http://www.imgt.org/ligmdb/view?id=J00228

TABLE 5 Amino acid residues of human kappa CL domain C-REGION Strands,EU turns and IMGT unique IGKC IMGT exon numbering loops for numberingfor amino acid numbering (107 aa) Kabat C-DOMAINs C-DOMAINs translation1-107 108-214 numbering 1.8 — — — — 1.7 — — — — 1.6 — — — — 1.5 — — — —1.4 (R) 1 108 108 1.3 T 2 109 109 1.2 V 3 110 110 1.1 A 4 111 111A-STRAND 1 A 5 112 112 2 P 6 113 113 3 S 7 114 114 4 V 8 115 115 5 F 9116 116 6 I 10 117 117 7 F 11 118 118 8 P 12 119 119 9 P 13 120 120 10 S14 121 121 11 D 15 122 122 12 E 16 123 123 13 Q 17 124 124 14 L 18 125125 15 K 19 126 126 AB-TURN 15.1 — — — — 15.2 — — — — 15.3 — — — —B-STRAND 16 S 20 127 127 17 G 21 128 128 18 T 22 129 129 19 A 23 130 13020 S 24 131 131 21 V 25 132 132 22 V 26 133 133 23 C 27 134 134 24 L 28135 135 25 L 29 136 136 26 N 30 137 137 BC-LOOP 27 N 31 138 138 28 F 32139 139 29 Y 33 140 140 30 P 34 141 141 31 — — — — 34 — — — — 35 R 35142 142 36 E 36 143 143 37 A 37 144 144 38 K 38 145 145 C-STRAND 39 V 39146 146 40 Q 40 147 147 41 W 41 148 148 42 K 42 149 149 43 V 43 150 15044 D 44 151 151 45 N 45 152 152 CD-STRAND 45.1 A 46 153 153 45.2 L 47154 154 45.3 Q 48 155 155 45.4 S 49 156 156 45.5 G 50 157 157 45.6 — — —— 45.7 — — — — D-STRAND 77 N 51 158 158 78 S 52 159 159 79 Q 53 160 16080 E 54 161 161 81 S 55 162 162 82 V 56 163 163 83 T 57 164 164 84 E 58165 165 DE-TURN 84.1 Q 59 166 166 84.2 D 60 167 167 84.3 S 61 168 16884.4 K 62 169 169 84.5 D 63 170 170 84.6 — — — — 84.7 — — — — 85.7 — — —— 85.6 — — — — 85.5 — — — — 85.4 S 64 171 171 85.3 T 65 172 172 85.2 Y66 173 173 85.1 S 67 174 174 E-STRAND 85 L 68 175 175 86 S 69 176 176 87S 70 177 177 88 T 71 178 178 89 L 72 179 179 90 T 73 180 180 91 L 74 181181 92 S 75 182 182 93 K 76 183 183 94 A 77 184 184 95 D 78 185 185 96 Y79 186 186 EF-TURN 96.1 — — — — 96.2 — — — — F-STRAND 97 E 80 187 187 98K 81 188 188 99 H 82 189 189 100 K 83 190 190 101 V 84 191 191 102 Y 85192 192 103 A 86 193 193 104 C 87 194 194 FG-LOOP 105 E 88 195 195 106 V89 196 196 107 T 90 197 197 108 H 91 198 198 109 Q 92 199 199 110 G 93200 200 111 — — — — 112 — — — — 113 L 94 201 201 114 S 95 202 202 115 S96 203 203 116 P 97 204 204 117 V 98 205 205 G-STRAND 118 T 99 206 206119 K 100 207 207 120 S 101 208 208 121 F 102 209 209 122 N 103 210 210123 R 104 211 211 124 G 105 212 212 125 E 106 213 213 126 C 107 214 214127 — — — 215 128 — — — 216 ¹http://www.imgt.org/ligmdb/view?id=J00241

TABLE 6 Amino acid residues of human lambda CL domain Strands, C-REGIONturns and IMGT unique IGLC IMGT exon loops for numbering for amino acidnumbering Kabat C-DOMAINs C-DOMAINs translation¹ 1-106 numbering 1.8 — —— 1.7 — — — 1.6 — — — 1.5 (G) 1  107A 1.4 Q 2 108 1.3 P 3 109 1.2 K 4110 1.1 A 5 111 A-STRAND 1 N 6 112 2 P 7 113 3 T 8 114 4 V 9 115 5 T 10116 6 L 11 117 7 F 12 118 8 P 13 119 9 P 14 120 10 S 15 121 11 S 16 12212 E 17 123 13 E 18 124 14 L 19 125 15 Q 20 126 AB-TURN 15.1 — — — 15.2— — — 15.3 — — — B-STRAND 16 A 21 127 17 N 22 128 18 K 23 129 19 A 24130 20 T 25 131 21 L 26 132 22 V 27 133 23 C 28 134 24 L 29 135 25 I 30136 26 S 31 137 BC-LOOP 27 D 32 138 28 F 33 139 29 Y 34 140 30 P 35 14131 — — — 34 — — — 35 G 36 142 36 A 37 143 37 V 38 144 38 T 39 145C-STRAND 39 V 40 146 40 A 41 147 41 W 42 148 42 K 43 149 43 A 44 150 44D 45 151 45 G 46 152 CD-STRAND 45.1 S 47 153 45.2 P 48 154 45.3 V 49 15545.4 K 50 156 45.5 A 51 157 45.6 — — — 45.7 — — — D-STRAND 77 G 52 15878 V 53 159 79 E 54 160 80 T 55 161 81 T 56 162 82 K 57 163 83 P 58 16484 S 59 165 DE-TURN 84.1 K 60 166 84.2 Q 61 167 84.3 S 62 168 84.4 N 63170 84.5 — — — 84.6 — — — 84.7 — — — 85.7 — — — 85.6 — — — 85.5 — — —85.4 N 64 171 85.3 K 65 172 85.2 Y 66 173 85.1 A 67 174 E-STRAND 85 A 68175 86 S 69 176 87 S 70 177 88 Y 71 178 89 L 72 179 90 S 73 180 91 L 74181 92 T 75 182 93 P 76 183 94 E 77 184 95 Q 78 185 96 W 79 186 EF-TURN96.1 — — — 96.2 — — — F-STRAND 97 K 80 187 98 S 81 188 99 H 82 189 100 R83 190 101 S 84 191 102 Y 85 192 103 S 86 193 104 C 87 194 FG-LOOP 105 Q88 195 106 V 89 196 107 T 90 197 108 H 91 198 109 E 92 199 110 — — — 111— — — 112 — — — 113 — — — 114 G 93 200 115 S 94 203 116 T 95 204 117 V96 205 G-STRAND 118 E 97 206 119 K 98 207 120 T 99 208 121 V 100 209 122A 101 210 123 P 102 211 124 T 103 212 125 E 104 213 126 C 105 214 127 S106 215 128 — — 216 ¹http://www.imgt.org/ligmdb/view?id=X51755

“Immunoglobulin Allotype”

As used herein the term “allotype” or “immunoglobulin allotype”indicates that the immunoglobulin chain or polypeptide is encoded by aparticular allele found in an individual. Different immunoglobulinallotypes have different sequences reflecting the natural variationfound in the alleles of an individual's genome. The natural variationnormally manifests in the constant region of the immunoglobulin heavyand light chain alleles and therefore different allotypes typicallydiffer in the constant region of the polypeptide, as defined above.

“Derived From”

As used herein the term “derived from” a designated protein (e.g. ahuman protein) refers to the origin of the polypeptide or amino acidsequence. In the context of the present invention, a fragment “derivedfrom” a designated protein means a fragment having an amino acidsequence which is essentially identical to the amino acid sequence ofthe originating or starting polypeptide. For example, a fragment derivedfrom a human immunoglobulin allotype will have an amino acid sequencecorresponding to the section of the human immunoglobulin polypeptidefrom which the fragment is taken.

In certain embodiments, the amino acid sequence which is derived from aparticular starting polypeptide is not contiguous. For example, in oneembodiment, one, two, three, four, five, or six CDRs are derived from astarting antibody. In one embodiment, the polypeptide or amino acidsequence which is derived from a particular starting polypeptide oramino acid sequence has an amino acid sequence that is essentiallyidentical to that of the starting sequence, or a portion thereof whereinthe portion consists of at least 3-5 amino acids, at least 5-10 aminoacids, at least 10-20 amino acids, at least 20-30 amino acids, or atleast 30-50 amino acids, or which is otherwise identifiable to one ofordinary skill in the art as having its origin in the starting sequence.In one embodiment, the one or more CDR sequences derived from thestarting antibody are altered to produce variant CDR sequences, e.g.affinity variants, wherein the variant CDR sequences maintain targetantigen binding activity.

“Specificity” and “Multispecific Antibody”

The term “specificity” refers to the ability of an antibody tospecifically bind (e.g., immunoreact with) a given target antigen, e.g.,a human target antigen. An antibody molecule may be monospecific andcontain one or more binding sites which specifically bind a singleepitope on a single target antigen. Alternatively, an antibody moleculemay be “multispecific” and contain two or more antigen binding siteswhich specifically bind different epitopes either within the sameantigen or located within different target antigens. For example, abispecific antibody has two antigen binding sites capable of recognisingand binding two different target epitopes or antigens. In order toachieve multiple specificities, multispecific antibodies are engineeredto include different combinations or pairings of heavy and light chainpolypeptides with different VH-VL pairs.

“Chimeric”

A “chimeric” protein comprises a first amino acid sequence linked to asecond amino acid sequence with which it is not naturally linked innature. The amino acid sequences may normally exist in separate proteinsthat are brought together in the fusion polypeptide or they may normallyexist in the same protein but are placed in a new arrangement in thefusion polypeptide. A chimeric protein may be created, for example, bychemical synthesis, or by creating and translating a polynucleotide inwhich the peptide regions are encoded in the desired relationship.Exemplary chimeric antibodies include fusion proteins comprising VHand/or VL domains derived from one species e.g. a species of the camelidfamily (or humanised variants thereof) fused to the constant domains ofa human antibody, e.g. human IgG1, IgG2, IgG3 or IgG4.

“Conservative Amino Acid Substitution”

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, an amino acid residue in animmunoglobulin polypeptide may be replaced with another amino acidresidue from the same side chain family. In another embodiment, a stringof amino acids can be replaced with a structurally similar string thatdiffers in order and/or composition of side chain family members.

“Engineered”

As used herein the term “engineered” includes manipulation of nucleicacid or polypeptide molecules by synthetic means (e.g. by recombinanttechniques, in vitro peptide synthesis, by enzymatic or chemicalcoupling of peptides or some combination of these techniques). Theantibodies of the invention may be engineered as described herein belowto incorporate variation into the region of the antibody moleculeoutside of the antigen binding site(s). The antibodies may also beengineered, for example, humanized and/or chimeric antibodies, andantibodies which have been engineered to improve one or more propertiessuch as antigen binding, stability/half-life or effector function.

“Humanising Substitutions”

As used herein, the term “humanising substitutions” refers to amino acidsubstitutions in which the amino acid residue present at a particularposition in the VH or VL domain antibody (for example a camelid-derivedantibody) is replaced with an amino acid residue which occurs at anequivalent position in a reference human VH or VL domain. The referencehuman VH or VL domain may be a VH or VL domain encoded by the humangermline. Humanising substitutions may be made in the framework regionsand/or the CDRs of an antibody, defined herein.

“Humanised Antibody or Variant”

As used herein the term “humanised antibody” or “humanised variant”refers to a variant antibody which contains one or more “humanisingsubstitutions” compared to a reference antibody, wherein a portion ofthe reference antibody (e.g. the VH domain and/or the VL domain or partsthereof containing at least one CDR) has an amino acid derived from anon-human species, and the “humanising substitutions” occur within theamino acid sequence derived from a non-human species.

“Heavy-Chain Only Antibody” or “VHH Antibody”

As used herein, the term “heavy-chain only antibody” or “VHH antibody”refers to a second type of antibody produced only by species of theCamelidae family, which includes camels, llama, alpaca. Heavy chain-onlyantibodies are composed of two heavy chains and are devoid of lightchains. Each heavy chain has a variable domain at the N-terminus, andthese variable domains are referred to as “VHH” domains in order todistinguish them from the variable domains of the heavy chains of theconventional heterotetrameric antibodies i.e. the VH domains, describedabove.

B. Multispecific Antibodies

The present invention relates to multispecific antibodies having bindingspecificity for multiple distinct epitopes, wherein “multiple” means twoor more distinct epitopes. The multispecific antibodies of the presentinvention may have binding specificity for at least two, at least three,at least four, at least five epitopes either within the same targetantigen or in different target antigens. In preferred embodiments, theantibodies of the invention are bispecific antibodies having specificityfor two distinct epitopes either within the same target antigen or indifferent target antigens.

First and Second Heavy Chain-Light Chain Pairings Define First andSecond Antigen Binding Sites

In order to achieve a multispecific binding profile, multispecificantibodies incorporate different heavy chain-light pairings orcombinations. The term “pairing” as used herein refers to the pairedconfiguration of heavy and light chains within the immunoglobulinmolecule, for example as shown in FIG. 2 , wherein each heavychain-light chain pair (or pairing) forms a variable region consistingof the VH and VL domains of the heavy and light chains, respectively. Itis these variable regions of the antibody molecule and in particular,the CDR sequences contained within each of the VH and VL domains thatdefine the antigen binding sites.

Multispecific antibodies of the present invention comprise or consist ofa first pairing between a first heavy chain and a first light chain anda second pairing between a second heavy chain and a second light chain.The corresponding heavy and light chains may pair via a disulphide bondas found in naturally occurring immunoglobulin molecules, although inthe context of the present invention, “pairing” may be association ofthe chains via any suitable means including non-covalent association.

The first and second heavy chain-light chain pairings create twodistinct variable regions within the antibody such that the antibody hasat least two distinct antigen binding sites capable of recognising andbinding different epitopes on the same or different antigens. It is thusthe precise pairing of the correct heavy and light chain combinationswithin the overall quaternary structure of the antibody molecule thatensures that the multispecific antibody has the correct binding profile.Mispairing between the respective heavy and light chains of the antibodywill result in an antibody either lacking the multispecific propertiesrequired and/or an immunoglobulin lacking antigen binding activityaltogether.

Selective Recognition Sites Outside the Antigen Binding Sites

The present invention addresses the problem of heavy chain-light chainmispairing by providing multispecific antibodies comprising orconsisting of first and second heavy chain-light chain pairings, whereinthe pairings can be distinguished by distinct or unique “selectiverecognition sites” located outside the antigen binding sites of themolecule. The “selective recognition sites” are distinct or unique inthe sense that they can be selectively recognised or selectively boundby different “selective recognition agents”, wherein such agents arecharacterised further below in the context of the methods of the presentinvention. The selective binding of “selective recognition agents” totheir cognate “selective recognition sites” allows the first and secondselective recognition sites to be distinguished or differentiated fromeach other. The first and second selective recognition sites are thusdifferentially bound by their respective selective recognition agents.

A selective recognition site according to the present invention is asite in the antibody molecule comprising or consisting of a distinct orunique combination of amino acid residues formed by a distinct heavychain-light chain pairing. As described below, each selectiverecognition site comprises or consists of at least one amino acidresidue from the heavy chain and at least one amino acid residue fromthe light chain of each pairing. For sites comprising more than oneamino acid residue from the heavy chain and/or more than one amino acidfrom the light chain, the residues from the same immunoglobulin chainmay be contiguous or non-contiguous in the polypeptide sequence. Thecombination of amino acid residues of each selective recognition sitetypically form a conformational epitope that can be selectivelyrecognised by a cognate selective recognition agent. The amino acidresidues that define the selective recognition site or conformationalepitope are the residues that contribute to the binding interfacebetween the site/epitope and the recognition agent that binds to thesite/epitope.

Each selective recognition site must comprise or consist of enough aminoacid residues and in particular, enough different amino acid residues,for it to be bound exclusively or at least preferentially by a selectiverecognition agent as described further below. The combination of aminoacid residues that form each selective recognition site will typicallybe clustered i.e. in close spatial proximity, in the overall quaternarystructure of the antibody molecule so as to form a conformationalepitope selectively recognised by a selective recognition agent.

Multispecific antibodies of the present invention include at least twoselective recognition sites: a first selective recognition site formedby the first heavy chain-light chain pairing and a second selectiverecognition site formed by the second heavy chain-light chain pairing.Each of the first and second selective recognition sites comprises atleast one amino acid residue from the heavy chain of the pair and atleast one amino acid residue from the light chain of the pair. Incertain embodiments, one or both first and second selective recognitionsites comprise at least two, at least three, at least four, at leastfive amino acid residues from the heavy chain of the pair and/or atleast two, at least three, at least four, at least five amino acidresidues from the light chain of the pair. Amino acid residues from boththe heavy chain and light chain of each pair contribute to theirrespective selective recognition sites as this allows for antibodieshaving the correct heavy chain-light chain pairings to be detected andisolated using the methods described herein.

In order for the first and second selective recognition sites to bedistinguished from each other and potentially from other recognitionsites located within the same antibody molecule, each selectiverecognition site must include enough amino acid residues for it to berecognised and bound by a selective recognition agent. Each selectiverecognition site may comprise or consist of a total of at least 2, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10 residues wherein at least one residue is from theheavy chain and at least one residue is from the light chain of eachpairing.

Each selective recognition site must include enough amino acid residuesthat differ, as compared with other recognition sites within the samemolecule, in order for the individual sites to be selectively recognisedi.e. distinguished from each other. In order for the first and secondselective recognition sites to be distinguished from each other, thecombination of amino acid residues of the first selective recognitionsite must differ by at least one amino acid residue from the combinationof amino acid residues of the second selective recognition site, and anyfurther sites within the same molecule. In certain embodiments, thecombination of amino acid residues of the first and second selectiverecognition sites differ by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15amino acid residues.

The amino acid residues that contribute to each selective recognitionsite may be located within any suitable part of the heavy chain and anysuitable part of the light chain, with the exception of the antigenbinding site. The term “antigen binding site” is defined elsewhereherein and contains residues from the CDR sequences of the VH and VLdomains.

In certain embodiments, the first and/or second selective recognitionsites comprise or consist of one or more residues located within theframework region of the VH domain of the heavy chain and/or one or moreresidues located within the framework region of the VL domain of thelight chain. In certain embodiments, one or both selective recognitionsites comprise one or more residues located within the framework regionsof the VH domain and/or the VL domain, preferably framework region 2(FR2) of the VH domain and/or the VL domain.

In preferred embodiments of the invention, the first and/or secondselective recognition site comprises or consists of one or more residueslocated within any suitable part of the constant region of the lightchain and one or more residues located within any suitable part of theconstant region of the heavy chain, wherein the constant region of theheavy chain is defined by the CH1-hinge-CH2-CH3 region of the molecule.Since each selective recognition site must include at least one residuecontributed from the heavy chain and light chain of each pairing, theresidue(s) from the heavy chain and the residue(s) from the light chainare preferably in close spatial proximity in the three-dimensionalpairwise configuration of the antibody. In certain embodiments, theresidue(s) from the heavy chain and the residue(s) from the light chainare within a distance of from about 20 Å to about 100 Å or from about 20Å to about 50 Å or from about 20 Å to about 25 Å in thethree-dimensional pairwise configuration of the antibody.

In certain embodiments, the first selective recognition site willcomprise or consist of one or more amino acid residues from the CLdomain of the first light chain and one or more residues from the CH1domain of the first heavy chain. Alternatively or in addition, thesecond selective recognition site will comprise or consist of one ormore amino acid residues from the CL domain of the second light chainand one or more amino acid residues from the CH1 domain of the secondheavy chain.

The selective recognition sites may comprise or consist of one or moresolvent-exposed residues located within the heavy chain and/or lightchain of the pairing. Solvent-exposed amino acids can be identifiedusing the program NACCESS as described by Rothlisberger et al. (J. Mol.Biol. (2005) 347, 773-789), the contents of which are incorporatedherein in their entirety, and on the Plueckthun website.

As explained above, the combination of amino acid residues of eachselective recognition site must differ by at least one amino acidresidue from the combination of amino acid residues of any otherrecognition sites within the same molecule in order for each selectiverecognition site to be distinguished by the binding of a selectiverecognition agent. The residues that differ between first and secondselective recognition sites may be contributed from the heavy chain ofthe pair and/or the light chain of the pair.

In certain embodiments, the multispecific antibodies of the inventioncomprise first and second heavy chains wherein the amino acid sequenceoutside of the antigen binding site differs by at least one amino acidresidue, and the first and/or second selective recognition sitecomprises or consists of one or more of the residues that differ betweenthe chains.

In the context of the present invention, amino acid residues are held todiffer if a comparison between two heavy chain polypeptides or two lightchain polypeptides identifies a different residue at a correspondingposition in the two chains.

In certain embodiments, the multispecific antibodies comprise first andsecond heavy chains wherein the amino acid sequences of the constantregions, and preferably the amino acid sequences of the CH1 domains,differ by at least one amino acid residue and the first and/or secondselective recognition site comprises or consists of one or more of theresidues that differ in the constant region, particularly in the CH1domain. In certain embodiments, the first and second heavy chains differby at least one amino acid residue in the solvent exposed regions of theheavy chains, the first and/or second selective recognition sitecomprises or consists of one of said solvent-exposed residues. Incertain embodiments, the first and second heavy chains differ by atleast one residue located outside of the binding interface between thepaired or dimerized CH3 domains of the first and second heavy chains andthe first and/or second selective recognition site comprises or consistsof at least one of said residues.

In certain embodiments, the first and second heavy chains differ by atleast one amino acid residue in the CH1 domain located outside of thebinding interface between the CH1 domain of the heavy chain and the CLdomain of the paired light chain and the first and/or second selectiverecognition site comprises or consists of one or more of said residues.Residues located at the CH1/CL interface are typically defined asresidues having a reduction in solvent accessibility of more than 40%when the CH1 domain and CL domain are paired (Rothlisberger et al. J.Mol. Biol. (2005) 347, 773-789). Tables 7 and 8 below identify theresidues in the CH1 domain of an IgG1 and the CL domain of a kappa lightchain, respectively, that exhibit a loss of more than 40% solventaccessibility when a CH1-CL domain pairing is formed. In certainembodiments, the first and second heavy chains differ by at least oneamino acid residue in the CH1 domain that is not listed in Table 7and/or the first and second light chains differ by at least one aminoacid residue in the CL domain that is not listed in Table 8 and thefirst and/or second selective recognition site comprises or consists ofone or more of said residues.

TABLE 7 Residues of CH1 buried in the CH1-CL domain interface (loss ofmore than 40% solvent accessibility when in complex) according toRothlisberger et al. (J. Mol. Biol. (2005) 347, 773-789) CH1 EU IMGTunique IGHG1 IMGT exon numbering numbering for amino acid numbering (98aa) Kabat C-domains translation¹ 1-98 118-215 numbering 5 F 9 126 122 6P 10 127 123 7 L 11 128 124 9 P 13 130 126 10 S 14 131 127 11 S 15 132128 14 T 18 135 133 20 A 24 141 139 21 L 25 142 140 22 G 26 143 141 24 L28 145 143 26 K 30 147 145 79 H 51 168 172 81 F 53 170 174 82 P 54 171175 84 V 56 173 177 84.2 Q 58 175 179 85.1 S 64 181 186 86 S 66 183 18888 V 68 185 190 119 K 96 213 221

TABLE 8 Residues of CL buried in the CH1-CL domain interface (loss ofmore than 40% solvent accessibility when in complex) according toRothlisberger et al. (J. Mol. Biol. (2005) 347, 773-789) C-REGION EUIMGT unique IGKC IMGT exon numbering numbering for amino acid numbering(107 aa) Kabat C-DOMAINs translation 1-107 108-214 numbering 5 F 9 116116 6 I 10 117 117 7 F 11 118 118 8 P 12 119 119 9 P 13 120 120 10 S 14121 121 12 E 16 123 123 13 Q 17 124 124 20 S 24 131 131 22 V 26 133 13324 L 28 135 135 26 N 30 137 137 27 N 31 138 138 79 Q 53 160 160 81 S 55162 162 83 T 57 164 164 84.2 D 60 167 167 85.1 S 67 174 174 85 L 68 175175 86 S 69 176 176 88 T 71 178 178 90 T 73 180 180 121 F 102 209 209126 C 107 214 214

Alternatively or in addition, the multispecific antibodies of theinvention may comprise first and second light chains wherein the aminoacid sequence outside of the antigen binding site differs by at leastone amino acid residue and the first and/or second selective recognitionsite comprises or consists of one or more of the residues that differbetween the chains. In certain embodiments, the multispecific antibodiescomprise first and second light chains wherein the amino acid sequencesof the constant regions i.e. the CL domains, differ by at least oneamino acid residue and the first and/or second selective recognitionsite comprises one or more of the residues that differ in the CL domain.In certain embodiments, the first and second light chains differ by atleast one amino acid residue in the CL domain located outside of theinterface between the CL domain of the light chain and the CH1 domain ofthe paired heavy chain and the first and/or second selective recognitionsite comprises or consists of one or more of said residues.

In preferred embodiments, the multispecific antibodies comprise firstand second heavy chains that differ by at least one amino acid residueoutside of the antigen binding sites and first and second light chainsthat differ by at least one amino acid residue outside of the antigenbinding sites and the first and/or second selective recognition sitescomprise or consist of residues that differ between both the heavychains and the light chains. In particularly preferred embodiments, thefirst and second heavy chains differ by at least one amino acid residuein the CH1 domain and the first and second light chains differ by atleast one amino acid residue in the CL domain and the first and/orsecond selective recognition sites comprise or consist of residues thatdiffer between the CH1 domains of the heavy chains and the CL domains ofthe light chains. In particularly preferred embodiments, the residue(s)that differ between the first and second heavy chains and the residue(s)that differ between the first and second light chains are locatedoutside of the binding interface between the CL domain and CH1 domain.

Naturally Occurring Variation in Immunoglobulin Heavy and Light Chains

One of the advantages of the present invention is that multispecificantibodies having at least two distinct selective recognition sites asdefined above can be generated using combinations of naturally occurringheavy and light chains, and in particular heavy chains and light chainscomprising at least the constant region of naturally occurring heavy andlight chains. This minimises the need for extensive protein engineeringand ensures that any unfavourable properties associated with mutatedimmunoglobulin sequences e.g. unwanted immunogenicity can be avoided.Furthermore, the use of naturally occurring heavy chains or fragmentsthereof in multispecific antibodies according to the present inventionhelps to preserve the natural effector function of the antibodies.

Heavy Chains

In certain embodiments, the multispecific antibodies as described hereincomprise a first pairing comprising a first heavy chain of a particularimmunoglobulin subtype and a second pairing comprising a second heavychain of a different immunoglobulin subtype. In particular, providedherein are multispecific antibodies wherein at least the constant regionof the first heavy chain and at least the constant region of the secondheavy chain are derived from different immunoglobulin subtypes. The term“immunoglobulin subtype” is as defined elsewhere herein and is usedinterchangeably with the terms “immunoglobulin subclass” and“immunoglobulin isotype”.

The different immunoglobulin subtypes are preferably from the samespecies and are preferably human. In a particular embodiment, thepresent invention provides a multispecific antibody comprising first andsecond heavy chain-light chain pairings as defined above wherein thefirst and second heavy chain each comprise the constant region of adifferent human immunoglobulin subtype and the different subtypes differby at least one amino acid residue in the constant region, preferably inthe CH1 domain.

In certain embodiments, the different immunoglobulin subtypes areselected from IgG1, IgG2, IgG3 and IgG4, preferably IgG1, IgG2, IgG3 andIgG4 of human origin. Polypeptide chains of different immunoglobulinsubtypes, for example IgG1 and IgG2, differ in their amino acid sequenceoutside of the antigen binding site including in the constant region.Therefore, in embodiments wherein the first and second heavy chains aredifferent immunoglobulin subtypes or comprise at least the constantregion thereof, the first and/or second selective recognition sites cancomprise or consist of one or more of the amino acid residues thatdiffer at the corresponding position between the different subtypes. Incertain embodiments, the first and/or second selective recognition sitescomprise or consist of one or more of the amino acid residues thatdiffer between the CH1 domains of the different immunoglobulin subtypes.

The present invention can also exploit the variation in the amino acidsequences of different immunoglobulin heavy chain allotypes,particularly human heavy chain allotypes. Therefore, in certainembodiments, the multispecific antibodies as provided herein comprise afirst pairing with a first heavy chain of a particular immunoglobulinallotype and a second pairing with a second heavy chain of a differentimmunoglobulin allotype. In particular, provided herein aremultispecific antibodies comprising first and second heavy chain-lightchain pairings as defined above wherein at least the constant region ofthe first heavy chain and at least the constant region of the secondheavy chain are derived from different immunoglobulin allotypes and thedifferent allotypes differ by at least one amino acid residue in theconstant region, preferably in the CH1 domain.

In preferred embodiments, the first and second heavy chains aredifferent human immunoglobulin allotypes or comprise at least theconstant region of different human immunoglobulin allotypes. Forexample, the first and second heavy chains may comprise at least theconstant region of different human immunoglobulin allotypes selectedfrom: IGGH1.1, IGGH1.3, IGGH2, IGGH2.2, IGGH2.4, IGGH4, IGGH3, IGG3.17and IGGH3.18.

Similar to the situation concerning multispecific antibodies comprisingheavy chains of different immunoglobulin subtypes, in embodimentswherein the first and second heavy chains are different immunoglobulinallotypes or comprise at least the constant region of differentimmunoglobulin allotypes, the first and/or second selective recognitionsites may comprise or consist of one or more amino acid residues thatnaturally differ at the corresponding position between the differentallotypes. In preferred embodiments, the first and/or second selectiverecognition site comprises or consists of one or more amino acidresidues within the CH1 domain that differs at the correspondingposition between the different immunoglobulin allotypes.

The sequences of the CH1 domains found within some of the differenthuman IgG heavy chain allotypes are shown in Table 9 and an alignment ofthe sequences is shown in FIG. 3 with natural variation indicated atamino acid positions 131, 133, 137, 138, 178, 189, 192, 193, 196, 199,203 and 214 of the sequences (based on EU numbering).

TABLE 9 CH1 sequences of different human immunoglobulin allotypesAllotype CH1 amino acid sequence SEQ ID NO. IGGH1.1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT 1FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV IGGH1.3ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT 2FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV IGGH2ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT 3FPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERK IGGH2.2ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT 4FPAVLQSSGLYSLSSVVTVTSSNFGTQTYTCNVDHKPSNTKVDKTVERK IGGH2.4ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT 5FPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNTKVDKTVERK IGGH4ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT 6FPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESK IGGH3ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT 7FPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPL GDTTHT IGG3.17ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT 8FPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVNHKPSNTKVDKRVELKTPL GDTTHT IGGH3.1ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT 9FPAVLQYSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPL GDTTHT

The multispecific antibodies of the invention may comprise or consist offirst and second heavy chain amino acid residues selected from thefollowing:

-   -   (i) at position 131: C in the first heavy chain; S in the second        heavy chain; and/or    -   (ii) at position 133: R in the first heavy chain; K in the        second heavy chain; and/or    -   (iii) at position 137: E in the first heavy chain; G in the        second heavy chain; and/or    -   (iv) at position 138: S in the first heavy chain; G in the        second heavy chain; and/or    -   (v) at position 178: S in the first heavy chain; Y in the second        heavy chain; and/or    -   (vi) at position 192: N in the first heavy chain; S in the        second heavy chain; and/or    -   (vii) at position 193: F in the first heavy chain; L in the        second heavy chain.

In preferred embodiments, multispecific antibodies of the inventioncomprise or consist of a first heavy chain comprising or consisting of Eat position 137 and S at position 138, and a second heavy chaincomprising or consisting of G at position 137 and Gat position 138. Inpreferred embodiments, multispecific antibodies of the inventioncomprise or consist of a first heavy chain comprising or consisting of Nat position 192 and F at position 193, and a second heavy chaincomprising or consisting of S at position 192 and L at position 193.

Exemplary bispecific antibodies of the invention are shown in FIG. 7 .The differences in the CH1 domains of the antibodies are shown incombination with first and second lambda and kappa light chains.However, it will be appreciated that the lambda and kappa chains couldbe reversed such that the different CH1 domains are paired with a lightchain of the opposite class. For example, in the first embodiment shown,the CH1 domain comprising the C131 residue is shown paired with aClambda domain and the S131 residue is shown paired with a Ckappadomain. However, this pairing could be reversed such that the CH1 domaincomprising the C131 residue is paired with the Ckappa domain and the CH1domain comprising the S131 residue is paired with the Clambda domain.

In certain embodiments, wherein multispecific antibodies of theinvention comprise a first heavy chain and a different second heavychain, each having at least a constant region selected from the humanimmunoglobulin allotypes: IGGH1.1, IGGH1.3, IGGH2, IGGH2.2, IGGH2.4,IGGH4, IGGH3, IGG3.17 and IGGH3.18, the first and/or second selectiverecognition sites may comprise one or more residues selected frompositions: 131, 133, 137, 138, 178, 189, 192, 193, 196, 199, 203 and 214of the CH1 domain, preferably positions 131, 133, 137, 138, 178, 192 and193 of the CH1 domain.

The amino acid residues of the heavy chain constant region in closespatial proximity to the constant region of the paired light chain arepreferred residues for contributing to the selective recognition site.Exemplary residues are indicated in the space fill model shown in FIG. 4. In certain embodiments, the first and second selective recognitionsites, respectively, comprise or consist of heavy chain residuesselected from the following:

-   -   (i) at position 131: C in the first heavy chain; S in the second        heavy chain; and/or    -   (ii) at position 133: R in the first heavy chain; K in the        second heavy chain; and/or    -   (iii) at position 137: E in the first heavy chain; G in the        second heavy chain; and/or    -   (iv) at position 138: S in the first heavy chain; G in the        second heavy chain; and/or    -   (v) at position 178: S in the first heavy chain; Y in the second        heavy chain; and/or    -   (vi) at position 192: N in the first heavy chain; S in the        second heavy chain; and/or    -   (vii) at position 193: F in the first heavy chain; L in the        second heavy chain.

In preferred embodiments, the first and second selective recognitionsites, respectively, comprise or consist of combinations of heavy chainresidues selected from the following:

-   -   (i) E at position 137 and S at position 138 in the first heavy        chain and G at position 137 and G at position 138 in the second        heavy chain; and/or    -   (ii) N at position 192 and F at position 193 in the first heavy        chain and S at position 192 and L at position 193 in the second        heavy chain.

As noted above, exemplary bispecific antibodies of the invention areshown in FIG. 7 and the residues indicated in the CH1 domains thatdiffer between the heavy chains may contribute to the first and/orsecond selective recognition sites, respectively.

The positions of the amino acid residues in the heavy chains areidentified above with reference to the EU numbering scheme as applied tohuman IgG1 molecules (see Table 2). It would be within the capabilitiesof the skilled person to align the amino acid sequence of a human IgG1heavy chain with the amino acid sequence of a different heavy chain inorder to identify equivalent or corresponding residues. These equivalentor corresponding residues are within the scope of the present invention.

Light Chains

Alternatively or in addition to naturally occurring heavy chains,multispecific antibodies of the invention may comprise first and secondlight chains of different immunoglobulin classes or differentimmunoglobulin allotypes. In particular, provided herein aremultispecific antibodies wherein at least the constant region of thefirst light chain and at least the constant region of the second lightchain are derived from different immunoglobulin classes or allotypes.

In certain embodiments, multispecific antibodies of the inventioncomprise a lambda light chain as the first light chain and a kappa lightchain as the second light chain or a kappa light chain as the firstlight chain and lambda light chain as the second light chain. The lambdaand/or kappa light chains, or at least the constant regions thereof, arepreferably human lambda and/or kappa light chains. In embodimentswherein the antibody comprises a first lambda light chain and a secondkappa light chain, the first selective recognition site may comprise oneor more residues specific to the lambda light chain and/or the selectiverecognition site may comprise one or more residues specific to the kappalight chain. As used herein, a residue “specific” to the kappa or lambdalight chain is a residue located at a position within the polypeptideamino acid sequence that differs from the amino acid residue located atthe corresponding position within the polypeptide sequence of the otherclass of light chain.

As an alternative to antibodies comprising light chains of differentclasses, the multispecific antibodies as provided herein may comprise afirst pairing having a first light chain of a particular immunoglobulinallotype and a second pairing having a second light chain of a differentimmunoglobulin allotype. In certain embodiments, at least the constantregion of the first light chain and at least the constant region of thesecond light chain are derived from different immunoglobulin allotypes.In preferred embodiments, the first and second light chains aredifferent human immunoglobulin allotypes, optionally selected fromdifferent kappa or lambda allotypes. For example, the first and secondlight chains or at least the constant regions thereof may be differenthuman immunoglobulin allotypes selected from the human lambda chainallotypes: IGLC1*01, IGLC2*01, IGLC3*01, IGLC7*01, IGLC6*01.

As described above for multispecific antibodies comprising heavy chainsof different allotypes, in embodiments wherein the first and secondlight chains are different immunoglobulin allotypes or comprise at leastthe constant region of different immunoglobulin allotypes, the firstand/or second selective recognition sites may comprise or consist of oneor more amino acid residues that naturally differ at the correspondingposition between the different allotypes. In preferred embodiments, thefirst and/or second selective recognition site comprises or consists ofone or more amino acid residues within the CL domain that differsbetween the different immunoglobulin allotypes. The sequences of the CLdomains found within some of the different human lambda chain allotypesare shown in Table 10 and an alignment of the sequences is shown in FIG.5 .

TABLE 10 CL sequences of different human lambda immunoglobulin allotypesAllotype CL amino acid sequence SEQ ID NO. IGLC1*01PKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKA 10GVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTV APTECS IGLC2*01GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPV 11KAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEK TVAPTECS IGLC3*01QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVK 12AGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKT VAPTECS IGLC7*01GQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSPV 13KVGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCRVTHEGSTVEK TVAPAECS IGLC6*01GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVKVAWKADGSPV 14NTGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEK TVAPAECS

The multispecific antibodies of the invention may comprise or consist offirst and second light chain amino acid residues selected from thefollowing:

-   -   (i) at position 112: N in the first light chain; A in the second        light chain; and/or    -   (ii) at position 114: T in the first light chain; S in the        second light chain; and/or    -   (iii) at position 212: T in the first light chain; A in the        second light chain.

In certain embodiments, wherein multispecific antibodies of theinvention comprise a first light chain and a different second lightchain, each selected from the human immunoglobulin allotypes: IGLC1*01,IGLC2*01, IGLC3*01, IGLC7*01, IGLC6*01, the first and/or secondselective recognition sites may comprise or consist of one or moreresidues selected from positions 112, 114 and 212. As shown in the spacefill model in FIG. 6 , these residues are in close spatial proximity tothe CH1 domain of the paired heavy chain and are preferred residues forcontributing to the selective recognition site.

In certain embodiments, the first and second selective recognitionsites, respectively, include light chain residues selected from thefollowing:

-   -   (i) at position 112: N in the first light chain; A in the second        light chain; and/or    -   (ii) at position 114: T in the first light chain; S in the        second light chain; and/or    -   (iii) at position 212: T in the first light chain; A in the        second light chain.

The positions of the amino acid residues in the light chain areidentified with reference to the numbering scheme developed by Kabat etal. as applied to human lambda light chains (see Table 6). It would bewithin the capabilities of the skilled person to align the amino acidsequence of a human lambda light chain with the amino acid sequence of adifferent light chain in order to identify equivalent or correspondingresidues. These equivalent or corresponding residues are within thescope of the present invention.

In preferred embodiments of the present invention, the multispecificantibody comprises a first heavy chain comprising at least the constantregion of a particular immunoglobulin allotype, preferably a humanallotype, and a second heavy chain comprising at least the constantregion of a different immunoglobulin allotype, preferably a humanallotype, wherein the allotypes differ by at least one amino acidresidue in the CH1 domain, and further comprises a lambda light chain,wherein at least the constant region is preferably human, as the firstlight chain and a kappa light chain, wherein at least the constantregion is preferably human, as the second light chain. In suchembodiments, the first and/or second selective recognition sitescomprise or consist of one or more residues that differ between theheavy chain allotypes, preferably in the constant region, morepreferably in the CH1 domain, and one or more residues that are specificto the lambda/kappa light chains, preferably in the CL domain.

Modified Antibodies

The multispecific antibodies of the present invention can be modified,as compared with naturally occurring immunoglobulin molecules, in anumber of different ways. Described below are various modifications andmodified antibody forms/structures that are applicable to multispecificantibodies according to all aspects of the invention described herein.

Antibodies Incorporating Fragments and/or Amino Acid Substitutions fromNaturally Occurring Immunoglobulin Chains

In addition to naturally occurring immunoglobulin chains and constantregions derived therefrom, the multispecific antibodies of the presentinvention may include immunoglobulin chains incorporating fragmentsderived from different naturally occurring immunoglobulin chains and/orimmunoglobulin chains from different species. As used herein, a“fragment derived from” a particular polypeptide is a length of aminoacids with a sequence identical to or substantially identical to asequence of amino acids in the originating polypeptide.

The immunoglobulin chains of the multispecific antibodies describedherein may be full length as compared with naturally occurringimmunoglobulin heavy and/or light chains and “incorporate a fragment”such that a region of the full length immunoglobulin chain backbone issubstituted with the fragment. The fragment will typically correspond tothe region equivalent to the substituted region located within theoriginating polypeptide. The immunoglobulin chains of the multispecificantibodies described herein may thus be composite immunoglobulin chainsincorporating fragments derived from different immunoglobulin chainsinto a full-length polypeptide chain (see FIG. 8A).

Alternatively, the immunoglobulin chains of the multispecific antibodiesdescribed herein may be truncated, as described further below, andincorporate fragments shorter in length than the overall heavychain/light chain length. Such truncated chains may also be compositechains incorporating fragments derived from different immunoglobulinchains.

The multispecific antibodies of the invention may comprise first andsecond heavy chains wherein the first heavy chain comprises a fragmentderived from a particular immunoglobulin subtype or allotype and thesecond heavy chain comprises a corresponding fragment derived from thesame region of a different immunoglobulin subtype or allotype, includingbut not limited to the immunoglobulin subtypes and allotypes describedelsewhere herein. Alternatively or in addition, the multispecificantibodies of the present invention may comprise first and second lightchains wherein the first light chain comprises a fragment derived from aparticular immunoglobulin class (kappa or lambda) or allotype and thesecond light chain comprises a corresponding fragment derived from thesame region of a different immunoglobulin class (kappa or lambda) orallotype, including but not limited to the immunoglobulin allotypesdescribed elsewhere herein.

The position and length of each fragment may be selected such that itincludes at least some of the natural variation i.e. amino aciddifferences, found at particular positions of naturally occurringdifferent immunoglobulin classes, subtypes and allotypes. For example,the human IgG subtypes and allotypes shown in FIG. 3 exhibit variationat positions 131, 133, 137, 138, 178, 189, 192, 193, 196, 199, 203 and214. Therefore, a fragment incorporated into the first heavy chain maycomprise or consist of a region incorporating at least one of theresidues identified above derived from a first human IgG subtype orallotype and a fragment incorporated into the second heavy chain maycomprise or consist of a region incorporating at least one of theresidues identified above derived from a second human IgG subtype orallotype and having at least one amino acid difference located in thisregion.

An exemplary composite bispecific IgG antibody according to the presentinvention is shown schematically in FIG. 8A. This bispecific antibodyhas a first IgG heavy chain backbone incorporating a fragment derivedfrom the CH1 domain of a first human IgG allotype and a second IgG heavychain backbone incorporating a fragment derived from the CH1 domain of asecond human IgG allotype. The fragment incorporated into the firstchain includes residues E137 and S138 and the fragment incorporated inthe second chain includes residues G137 and G138. These differences inthe CH1 domains allow the first and second heavy chains to bedistinguished or differentiated from each other.

The first and second light chains may also include fragments asdescribed above, for example fragments derived from different humanallotypes. The human lambda allotypes shown in FIG. 5 exhibit variationi.e. amino acid differences, for example at positions 112 and 114.Therefore, a fragment incorporated into the first light chain maycomprise or consist of a region including one or both of these positionsfrom a first human lambda allotype and a fragment incorporated into thesecond light chain may comprise or consist of a region including one orboth of these positions from a second human lambda allotype whereinthere is at least one amino acid difference between the allotypes. Itwould be within the capabilities of a person skilled in the art toidentify suitable fragments from naturally occurring heavy and lightchain immunoglobulin classes, subtypes and/or allotypes whichincorporate similar amino acid sequence variation and engineermultispecific antibodies incorporating such fragments so as to generateantibodies having amino acid sequence variation outside of the antigenbinding site, preferably in the constant regions, between the first andsecond heavy chain-light chain pairings.

A fragment according to the present invention may have a length of atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10 residues wherein the position of thefragment is selected such that the corresponding fragment derived from adifferent immunoglobulin class, subtype or allotype has an amino acidsequence differing by at least one amino acid residue. In certainembodiments, a fragment derived from an immunoglobulin chain and inparticular, a human immunoglobulin chain may be at least 20, at least40, at least 50, at least 60, at least 70, at least 80, at least 90, atleast 100, at least 110, at least 120, at least 130, at least 140, atleast 150, at least 160, at least 170, at least 180, at least 190 or atleast 200 amino acids in length. A fragment may also comprise or consistof a complete domain derived from the immunoglobulin chain wherein thedomain is selected from the CH1, CH2 or CH3 domain of the heavy chain orthe CL domain of the light chain. In preferred embodiments, thefragments inserted into the heavy chains comprise or consist of theentire CH1 domain and the fragments inserted into the light chainscomprise or consist of the entire CL domain.

For antibodies of the invention comprising fragments that produce one ormore amino acid sequence differences between the first and second heavychains and/or the first and second light chains, the first and/or secondselective recognition sites may comprise one or more of the residuesthat differ between the fragments. Therefore, the introduction ofdistinct fragments from naturally occurring immunoglobulin heavy andlight chains can be used to create the distinct first and secondselective recognition sites within the multispecific antibodies of theinvention.

Alternatively, or in addition to the embodiments described above,multispecific antibodies according to the present invention may compriseimmunoglobulin chains including one or more amino acid substitutionsderived from different naturally occurring immunoglobulin chains and/orimmunoglobulin chains from different species. In particular,multispecific antibodies may comprise or consist of heavy chains and/orlight chains wherein a particular amino acid in the heavy and/or lightpolypeptide chain is substituted with a different amino acid that isfound at the corresponding position in a different immunoglobulin class,subtype or allotype.

The amino acid substitutions introduced into the heavy and/or lightchains of the multispecific antibodies of the present invention mayderive from any naturally occurring immunoglobulin chains, preferablyimmunoglobulin chains of human origin, including but not limited to theimmunoglobulin classes, subtypes and allotypes described elsewhereherein.

In certain embodiments, the multispecific antibodies of the inventioncomprise or consist of first and second heavy chains wherein the firstheavy chain comprises one or more amino acid substitution(s) derivedfrom a particular immunoglobulin subtype or allotype and the secondheavy chain comprises one or more amino acid substitution(s) from adifferent immunoglobulin subtype or allotype, wherein the substitutionscreate differences between the amino acid sequences of the first andsecond heavy chains. Preferably, the amino acid substitution(s) arelocated in the CH1 domains of the heavy chains. Alternatively or inaddition, the multispecific antibodies may comprise first and secondlight chains wherein the first light chain comprises one or more aminoacid substitution(s) derived from a particular immunoglobulin class(kappa or lambda) or allotype and the second light chain comprises oneor more amino acid substitution(s) derived from a differentimmunoglobulin class (kappa or lambda) or allotype, wherein thesubstitutions create differences between the amino acid sequences of thefirst and second light chains. Preferably, the amino acidsubstitution(s) are located in the CL domains of the light chains.

In certain embodiments, the heavy chains and/or light chains comprise orconsist of combinations of amino acid substitutions derived fromnaturally occurring immunoglobulin chains such that the first and secondheavy chains and/or first and second light chains differ in amino acidsequence at the substituted positions. A “combination” of amino acidsubstitutions may include at least 2, 3, 4, 5, 6, 7 amino acidsubstitutions, wherein the substitutions occur at contiguous ornon-contiguous locations in the polypeptide chains.

An exemplary bispecific IgG antibody according to the present inventionis shown schematically in FIG. 8B. This bispecific antibody has a firstIgG heavy chain including the amino acid substitutions R133, E137, S138,S178, N192, F193 and a second IgG heavy chain including the amino acidsubstitutions K133, G137, G138, Y178, S192, L193. These amino acidsubstitutions derive from the naturally occurring human IgG allotypesshown in FIG. 3 . Multispecific antibodies of the invention may compriseor consist of amino acid substitutions derived from any of theimmunoglobulin allotypes described elsewhere herein and in preferredembodiments include amino acid substitutions corresponding to the aminoacid differences between first and second heavy chains shown in FIG. 7 .

In certain embodiments, multispecific antibodies comprise or consist offirst and second heavy chain amino acid substitutions selected from thefollowing:

-   -   (i) at position 131: C in the first heavy chain; S in the second        heavy chain; and/or    -   (ii) at position 133: R in the first heavy chain; K in the        second heavy chain; and/or    -   (iii) at position 137: E in the first heavy chain; G in the        second heavy chain; and/or    -   (iv) at position 138: S in the first heavy chain; G in the        second heavy chain; and/or    -   (v) at position 178: S in the first heavy chain; Y in the second        heavy chain; and/or    -   (vi) at position 192: N in the first heavy chain; S in the        second heavy chain; and/or    -   (vii) at position 193: F in the first heavy chain; L in the        second heavy chain.

In preferred embodiments, multispecific antibodies comprise or consistof first and second heavy chain amino acid substitutions selected fromthe following:

-   -   (i) E at position 137 and S at position 138 in the first heavy        chain and G at position 137 and G at position 138 in the second        heavy chain;    -   (ii) N at position 192 and F at position 193 in the first heavy        chain and S at position 192 and L at position 193 in the second        heavy chain;    -   (iii) R at position 133, E at position 137, S at position 138, S        at position 178, N at position 192 and F at position 193 in the        first heavy chain and K at position 133, G at position 137, G at        position 138, Y at position 178, S at position 192 and L at        position 193 in the second heavy chain.

For antibodies of the invention comprising amino acid substitutions thatproduce one or more amino acid sequence differences between the firstand second heavy chains and/or the first and second light chains, thefirst and/or second selective recognition sites may comprise one or moreof the substituted residues. Therefore, the introduction of distinctamino acid substitutions from naturally occurring immunoglobulin heavyand light chains can be used to create the distinct first and secondselective recognition sites within the multispecific antibodies of theinvention.

Mutations Affecting the Selective Recognition Sites

Alternatively or in addition to the natural variation found in the aminoacid sequences of naturally occurring immunoglobulin heavy and lightchains, the heavy and/or light chains incorporated into themultispecific antibodies described herein may be mutated. In particular,mutations may be introduced into the first and/or second heavy chainoutside of the antigen binding site, preferably in the constant region,more preferably in the CH1 domain, so as to create at least onedifference or at least one further difference (in addition to anynatural variation) between the amino acid residues at the correspondingpositions of the amino acid sequences of the first and second heavychains. Alternatively or in addition, mutations may be introduced intothe first and/or second light chain outside of the antigen binding site,preferably in the constant region i.e. the CL domain, so as to create atleast one difference or at least one further difference (in addition toany natural variation) between the amino acid residues at thecorresponding positions of the first and second light chains. Thesemutations may be mutations not found in naturally occurringimmunoglobulin chains. In certain embodiments, the multispecificantibodies of the present invention comprise or consist of first andsecond heavy chains and/or first and second light chains wherein theamino acid differences between the heavy chains and/or the amino aciddifferences between the light chains represent a combination of naturalvariation between the amino acid sequences of the chains and thepresence of one or more amino acid substitutions or engineered mutationsnot found in naturally occurring immunoglobulin chains.

The mutations may be introduced in solvent-exposed regions of the heavyand/or light chains. Alternatively or in addition, the mutations may beintroduced in regions of the heavy and/or light chain outside of thebinding interface between the CH1 and CL domains of the heavychain-light chain pairing as defined elsewhere herein. Techniques forintroducing mutations into the polynucleotide sequences encoding theheavy chain and/or the light chain polypeptides are known in the art andcould readily be used by a person skilled in the art to produceantibodies according to the invention.

For embodiments in which the first and/or second heavy chain has beenmutated to produce at least one difference in the amino acid sequence ofthe heavy chains outside of the antigen binding site, preferably withinthe constant region, the first and/or second selective recognition sitemay comprise one or more of the amino acid residues that differ betweenthe heavy chains. Alternatively or in addition, for embodiments in whichthe first and/or second light chain has been mutated to create at leastone difference in the amino acid sequence of the light chains outside ofthe antigen binding site, preferably within the constant domain, thefirst and/or second selective recognition site may comprise one or moreof the amino acid residues that differ between the light chains. Inpreferred embodiments, the first and/or second selective recognitionsites comprise or consist of amino acid residues that differ betweenfirst and second heavy chains and first and second light chains.

In certain embodiments, the first and/or second selective recognitionsite comprises or consists of amino acid residues that differ betweenfirst and second heavy chains wherein the site comprises or consists ofat least one residue that differs due to natural variation between theimmunoglobulin chains and at least one residue that has been substitutedor mutated so as to create a difference between first and second heavychains. Alternatively or in addition, the first and/or second selectiverecognition sites may comprise or consist of amino acid residues thatdiffer between first and second light chains wherein the site comprisesor consists of at least one residue that differs due to naturalvariation between the immunoglobulin chains and at least one residuethat has been substituted or mutated so as to create a differencebetween first and second light chains.

Additional Features and Mutations

The heavy and light chain polypeptides of the multispecific antibodiesof the present invention may derive from any source including heavy andlight chain polypeptides of mouse, rat, rabbit, goat, hamster, chicken,monkey or human origin. In certain embodiments, the multispecificantibodies of the invention may be derived from the Camelidae family andin particular llama (Lama glama).

The multispecific antibodies of the present invention may incorporatechimeric, humanised or substantially humanised variants of antibodies(as defined elsewhere herein), wherein portions of the multispecificantibody, and in particular portions or regions of the differentimmunoglobulin chains, are derived from different sources. For example,multispecific antibodies of the present invention may comprise at leastone camelid derived domain, preferably a camelid-derived VH and/or VLdomain. The camelid-derived domain(s) may incorporate humanisingsubstitutions as described in WO2011/080350, the contents of which areincorporated herein in their entirety.

The present invention encompasses chimeric antibodies having VH and/orVL domains derived from any species including but not limited toantibodies of mouse, rat, rabbit, goat, hamster, chicken, monkey orhuman origin, and heavy chain and/or light chain constant regionsderived from antibodies of another species. In preferred embodiments,the VH and/or VL domains are camelid-derived and the constant regions ofthe heavy and/or light chains are substantially human.

The multispecific antibodies of the present invention may also beengineered so as to incorporate mutations or modifications which havepreviously been reported to address the problem of multispecificantibody chain mispairing. For example, the CH3 domains of the first andsecond heavy chains may be engineered to include the “knobs into holes”mutations reported in (Carter P. J. Immunol. Methods (2001) 248: 7-15),which promote correct heavy chain heterodimerization in the Fc region.The VH and VL domains and/or CH1 and CL domains may also be engineeredas described in Lewis et al. (Nature Biotechnology (2014) 32: 191-198)so as to promote the correct pairing between the heavy and light chainsof the antibody molecule.

The antibodies of the invention may further comprise additionalmodifications, for example modifications to improve properties such asantigen binding, effector function and/or stability/half-life and/orreduce unfavourable properties such as immunogenicity in a human host.The antibodies of the invention may comprise any of the mutations orcombinations of amino acid residues described in WO2006/130834 (thecontents of which are incorporated herein in their entirety). Theseinclude the combination of mutations: Lys433, Phe434, Tyr436 in the CH3domain of the heavy chain, which together can enhance the bindingaffinity of the antibody for the FcRn receptor and therefore can help toprolong the serum half life of the antibody.

The additional mutations or modifications described above, either toaddress the issue of chain mispairing or to alter the properties of theantibodies described herein, do not contribute to or affect theselective recognition sites.

Multispecific Antibodies Having Native Structure

Multispecific antibodies of the present invention may comprise fulllength heavy chains and/or full length light chains, wherein“full-length” is used herein to mean that the amino acid sequence of theimmunoglobulin chain is essentially identical in length to the length ofa naturally occurring heavy and/or light chain.

The antibodies may comprise first and second heavy chain-light chainpairings wherein the antigen binding site of each pairing recognises adifferent epitope (on the same or different antigens) and the antibodyis monovalent for each epitope. In preferred embodiments, the antibodiesas described herein are bispecific and/or have the standard domainstructure shown in FIG. 2 for bispecific antibodies having first andsecond heavy chain-light chain pairings i.e. a domain structure ofVH-CH1-hinge-CH2-CH3 for the first and second heavy chains, and a domainstructure of VL-CL for the first and second light chains. In furtherpreferred embodiments, the antibodies of the invention are IgG moleculesand/or have the structure of a native or naturally-occurring IgGmolecule.

Truncated Multispecific Antibodies

The present invention also covers truncated forms of multispecificantibodies wherein “truncated” is used herein to mean antibodies havingheavy chains and/or light chains that are not full length as comparedwith naturally occurring heavy and light immunoglobulin chains orpolypeptides. Multispecific antibodies according to the presentinvention are required to have a first heavy chain-light chain pairingand a second heavy chain-light chain pairing, wherein the pairings canbe detected by first and second selective recognition agents thatrecognise distinct first and second selective recognition sites outsideof the antigen binding site of each pairing. However, the heavy andlight chains need not be full length as compared with naturallyoccurring immunoglobulin chains, provided that they contain a VH or VLdomain capable of binding to a target antigen and a region or domain ofadequate length to contribute residues to the distinct selectiverecognition sites of the antibody molecules described herein. The scopeof the present invention therefore extends to any multispecific antibodyhaving the combination of a first heavy chain-light chain pairing andsecond heavy chain-light chain pairing wherein the four chains arerequired to assemble correctly in order to produce an antibody havingthe correct binding profile.

In certain embodiments, the multispecific antibodies comprise or consistof a first heavy chain-light chain pairing and a second heavychain-light chain pairing wherein the first and second heavy chains eachconsist of a VH domain and a CH1 domain and the first and second lightchains each consist of a VL domain and a CL domain and the heavy chainsassociate via any non-covalent means. In a preferred embodiment,multispecific antibodies of the present invention are F(ab′)₂ fragments,particularly F(ab′)₂ fragments wherein the first and second heavy chainsare modified by means permitting dimerization of the chains, for exampleby the formation of a leucine zipper.

C. Polynucleotides, DNA Constructs and Host Cells

The present invention also provides polynucleotides encoding themultispecific antibodies described herein, and expression vectorscomprising such polynucleotides operably linked to regulatory sequenceswhich permit expression of the multispecific antibodies in a host cellor cell-free expression system. Further provided are host cells orcell-free expression systems containing these expression vectors.

Polynucleotide molecules encoding the multispecific antibodies of theinvention include, for example, recombinant DNA molecules. The terms“nucleic acid”, “polynucleotide” or a “polynucleotide molecule” are usedherein interchangeably and refer to any DNA or RNA molecule, eithersingle- or double-stranded and, if single-stranded, the molecule of itscomplementary sequence. In discussing nucleic acid molecules, a sequenceor structure of a particular nucleic acid molecule may be describedherein according to the normal convention of providing the sequence inthe 5′ to 3′ direction. In some embodiments of the invention, nucleicacids or polynucleotides are “isolated.” This term, when applied to anucleic acid molecule, refers to a nucleic acid molecule that isseparated from sequences with which it is immediately contiguous in thenaturally occurring genome of the organism in which it originated. Forexample, an “isolated nucleic acid” may comprise a DNA molecule insertedinto a vector, such as a plasmid or virus vector, or integrated into thegenomic DNA of a prokaryotic or eukaryotic cell or non-human hostorganism.

An isolated polynucleotide (either DNA or RNA) may further represent amolecule produced directly by biological or synthetic means andseparated from other components present during its production. Forrecombinant production of a multispecific antibody according to theinvention, one or more recombinant polynucleotide(s) encoding themultispecific antibody may be prepared (using standard molecular biologytechniques) and inserted into a replicable vector for expression in achosen host cell, or a cell-free expression system. Suitable host cellsmay be prokaryote, yeast, or higher eukaryote cells, specificallymammalian cells. Expression vectors suitable for use in each of thesehost cells are also generally known in the art.

It should be noted that the term “host cell” generally refers to acultured cell line (prokaryote or eukaryote). Whole human beings intowhich an expression vector encoding a multispecific antibody accordingto the invention has been introduced are explicitly excluded from thedefinition of a “host cell”.

D. Methods for Isolating Multispecific Antibodies

In a further aspect, the present invention provides methods forisolating the multispecific antibodies described herein. All embodimentsdescribed above in connection with multispecific antibodies of thepresent invention are applicable to the methods of the invention. Themethods exploit the distinct selective recognition sites and/or distinctamino acid sequences comprised within each of the heavy chain-lightchain pairings of the antibody molecule. This allows for the isolationof only those multispecific antibodies comprising both of the correctfirst and second heavy chain-light chain pairings.

For multispecific antibodies having first and second heavy chains thateach comprise at least a fragment derived from the constant region ofdifferent human immunoglobulin subtypes or different allotypes, themethods as described herein can exploit the natural variation foundwithin the constant region of these naturally occurring heavy chains soas to distinguish between the first and second heavy chain-light chainpairings. This also allows for the isolation of only those multispecificantibodies comprising both of the correct first and second heavychain-light chain pairings.

Selective Recognition Agents

As discussed above, the first and second selective recognition sitescomprised within the first and second heavy chain-light chain pairingsare distinct in the sense that they can be selectively recognised andselectively or differentially bound by distinct first and secondselective recognition agents.

The selective recognition agents according to the present invention bindexclusively or at least exhibit preferential binding to their respectiveselective recognition site. The selective recognition agents preferablyhave at least 50× higher affinity, preferably at least 100× higheraffinity for their cognate selective recognition site as compared withany other site or selective recognition site located within the sameantibody molecule or in an antibody molecule formed from the mispairingof the respective heavy and light chains of the antibody of interest. Inorder for the methods of the invention to be used to isolatemultispecific antibodies having the correct first and second heavychain-light chain pairings, the selective recognition agents mustexhibit no or minimal cross-reactivity with other recognition siteswithin the same antibody molecule or in an antibody molecule formed fromthe mispairing of the heavy and light chains of the antibody ofinterest.

Selective recognition agents according to the present invention may takeany suitable form, provided that the agent is capable of distinguishingbetween first and second selective recognition sites. In certainembodiments, at least one of the first and second selective recognitionagents is a polypeptide having binding specificity for its cognateselective recognition site. Such polypeptides may be selected from, butnot limited to: peptides; helix bundles and Affibody molecules orbinding fragments thereof.

In certain embodiments, at least one of the first and second selectiverecognition agents is a conventional antibody or antigen bindingfragment thereof, wherein the term “conventional antibody” is usedherein to describe heterotetrameric antibodies containing heavy andlight immunoglobulin chains arranged according to the “Y” configurationshown in FIG. 2 . Such conventional antibodies may derive from anysuitable species including but not limited to antibodies of mouse, rat,rabbit, goat, hamster, chicken, monkey or human origin. In certainembodiments, the first and/or second selective recognition agents areconventional antibodies derived from a species of the Camelidae family,preferably llama (Lama glama).

The conventional antibodies for use as selective recognition agents inthe context of the present invention may include modified conventionalantibody variants, such as chimeric antibodies, humanised antibodies andsuch like. The first and/or second selective recognition agents may beantigen binding fragments of conventional antibodies wherein the term“antigen binding fragment” includes F(ab′)₂ fragments, Fab fragments, Fvfragments, sFv fragments and highly related molecules such as thosebased upon antibody domains which retain specific binding affinity (forexample, single domain antibodies).

Conventional antibodies are particularly suitable as selectiverecognition agents for use in accordance with the methods of the presentinvention because antibodies typically exhibit binding specificity foran epitope associated with a particular target antigen. In embodimentsof the present methods in which the first and/or second selectiverecognition agent is a conventional antibody, the target antigen is themultispecific antibody molecule of interest and the first and/or secondselective recognition site is a first and/or second “epitope”, which canbe selectively recognised by a particular antibody.

Alternatively, or in addition, at least one of the first and secondselective recognition agents may be a camelid-derived heavy chain onlyantibody or an antigen binding fragment thereof. Camelid heavy chainantibodies, also referred to herein as “VHH antibodies” represent asecond type of antibody naturally produced in camelids. Heavy chain onlyantibodies are so called because they are composed of two heavy chainsand are devoid of light chains (Hamers-Casterman, et al. Nature. 1993;363; 446-8). Each heavy chain has a variable domain at the N-terminus,and these variable domains are referred to in the art as “VHH” domainsin order to distinguish them from the variable domains of the heavychains of the conventional antibodies i.e. the VH domains. Similar toconventional antibodies, the VHH domains of the molecule confer antigenbinding specificity and therefore VHH antibodies or fragments such asisolated VHH domains, are suitable for use in accordance with themethods of the present invention as selective first and/or secondrecognition agents.

For embodiments wherein the first and second selective recognitionagents are selected from either conventional heterotetrameric antibodiesor VHH antibodies, antibodies specific for their respective selectiverecognition sites or “epitopes” may be generated or obtained by activeimmunization of a host species with a polypeptide comprising the firstor second selective recognition site i.e. the first or second epitope.For the production of conventional antibodies, the host species may beselected from any of the following: mouse, rat, rabbit, goat, hamster,chicken, monkey, or species of the family Camelidae. For the productionof VHH antibodies, any species from the family Camelidae, including lamaspecies, may be immunized with a polypeptide including the respectiveselective recognition site or epitope.

Methods of Isolation and/or Purification

The methods as described herein can be used for isolation of amultispecific antibody from any suitable sample. In certain embodiments,the methods are used for purification of a multispecific antibody havinga defined antigen binding profile from a crude preparation including theantibody of interest, for example a preparation obtained from arecombinant expression system. The methods are particularly useful forthe production of an antibody preparation enriched for the multispecificantibody of interest from a starting material comprising a mixture ofantibodies. In certain embodiments, the methods may be used to preparean enriched or a homogeneous preparation of a multispecific antibodyhaving a defined antigen binding profile from a mixture of antibodies,wherein the mixture contains antibodies having the antigen bindingprofile of interest and one or more different antibodies that do nothave the required antigen binding profile, or have no binding activityat all. In certain embodiments, the mixture of antibodies from which themultispecific antibody is isolated contains antibodies having variousdifferent pairings of the first and second heavy and light chains foundin the multispecific antibody of interest. However, within the mixtureare antibodies that either do not include the full complement of firstand second heavy and light chains such that they are not multispecific,or do not include the correct pairings of first and second heavy andlight chains. For example, the mixture may include antibodies having thefirst and second pairings as required but also antibodies having (i)first heavy chain paired with first light chain only; (ii) second heavychain paired with second light chain only; (iii) first heavy chainpaired with second light chain; and/or (iv) second heavy chain pairedwith first light chain (see for example, the mixture shown in FIG. 1 ).

In certain embodiments, the methods of the present invention can be usedto isolate or purify multispecific antibodies following recombinantexpression in any suitable expression system or host system as describedelsewhere herein. Recombinant expression of different heavy and lightchain polypeptides in the same expression system, for example the samehost cell or cell-free expression system, can lead to the production ofantibodies having incorrect chain pairings and therefore the methods ofthe present invention can be used to isolate only those multispecificantibodies having the correct heavy chain-light chain pairingsconferring the antigen binding profile of interest.

The methods of the present invention involve a two-step selectionprocess which allows for the isolation of multispecific antibodieshaving both the first and second heavy chain-light chain pairings andthereby having the desired antigen binding profile. This two-stepselection process is represented diagrammatically in FIG. 9 .

In the first step, the sample containing the multispecific antibody ofinterest or suspected of containing such multispecific antibody iscontacted with a first selective recognition agent under conditions thatpermit binding of the first selective recognition agent to the firstselective recognition site, if present. Any antibodies incorporating thefirst heavy chain-light chain pairing will be bound by the firstselective recognition agent and can therefore be separated from thesample. Antibodies bound to the first selective recognition agent can bereleased or dissociated using any suitable technique so as to generate asecond sample containing all antibodies having at least the first heavychain-light chain pairing.

It is possible that only some of the antibodies in the second samplecontain the second heavy chain-light chain pairing and therefore themethod requires a second step wherein the second sample is contactedwith a second selective recognition agent with specificity for thesecond selective recognition site comprised within the second heavychain-light chain pairing. Multispecific antibodies having the secondpairing will be selectively bound by the second selective recognitionagent and can therefore be separated from the second sample. Release ofthe bound antibodies will yield a preparation enriched for themultispecific antibody of interest having both first and second heavychain-light chain pairings and thus having the antigen binding profileof interest.

In particular embodiments of the methods described herein, themultispecific antibodies to be isolated comprise or consist of a firstheavy chain-light chain pairing and a second heavy chain pairing whereinthe first and second heavy chain differ by at least one amino acidresidue in the constant region. In particular, the first and secondheavy chains may each comprise at least a fragment derived from theconstant region of different human immunoglobulin subtypes or differentimmunoglobulin allotypes, as described elsewhere herein. Formultispecific antibodies of the invention having first and second heavychains with fragments derived from different immunoglobulin subtypes orallotypes, the selective recognition agents may differentially recognisefirst and second heavy chain-light chain pairings by selectively bindingto first and second sites within the constant regions of the first andsecond heavy chain-light chain pairings, respectively, wherein one orboth sites comprises at least one of the residues that differ betweenthe immunoglobulin subtypes or allotypes, preferably at least one of theresides that differ between the CH1 domains.

The selective recognition agents may be arranged according to anysuitable format to permit contact with the sample. In certainembodiments, the first and/or second selective recognition agents may beimmobilized, for example, by attachment to a solid support. The firstand/or second selective recognition agents may in particular, be fixedto a solid support to generate affinity chromatography columns to whichthe sample(s) can be applied.

In a particular embodiment of the method of the invention, the initialsample or mixture is applied to a first affinity chromatography columnloaded with the first selective recognition agent capable of binding thefirst selective recognition site in the multispecific antibody ofinterest. Once all of the sample has passed through the column, boundantibodies may be eluted using any suitable technique and/or elutionbuffer. The eluate from this first column is then applied to a secondaffinity chromatography column loaded with the second selectiverecognition agent capable of binding to the second selective recognitionsite in the multispecific antibody of interest. Once all of the eluatefrom the first column has been left to pass through the second column,the antibody bound to the second column may be eluted using any suitabletechnique or elution buffer. The eluate from the second column willcontain a preparation of antibodies enriched for multispecificantibodies having the desired binding specificity for at least first andsecond antigens via correct first and second heavy chain-light chainpairings.

The methods of the present invention may be employed in combination withother antibody purification or isolation methods in order to achieveincreased yields or higher purity yields of homogenous antibodypreparations. For example, the methods described herein may be precededby passing the sample through a protein A affinity column so as toselect for only properly assembled antibodies containing at least twoheavy and two light chains.

The multispecific antibodies and methods of the present inventionrepresent an improvement over the prior art multispecific antibodies andmethods for a number of reasons. Firstly, in contrast to approachesbased on the use of anti-idiotypic binding agents, the antibodies andmethods of the present invention allow for the isolation ofmultispecific antibodies having the correct heavy chain-light chainpairings using a universally applicable technique. In particular, thefact that the multispecific antibodies comprise at least first andsecond heavy chain-light chain pairings which can be distinguished onthe basis of distinct amino acid sequences in the form of distinctselective recognition sites located outside of the antigen binding siteallows for the isolation of any multispecific antibodies comprising atleast first and second selective recognition sites, irrespective of theantigen binding profile of the multispecific antibody. In particular,multispecific antibodies having binding specificity for any combinationof epitopes and target antigens can be engineered so as to containcommon first and second selective recognition sites within the heavychain-light chain pairings outside of the antigen binding sites. Itfollows that common selective recognition agents designed tospecifically recognise and bind particular first and second selectiverecognition sites may be developed and used to isolate and/or purifymultispecific antibodies having any number of different antigen bindingprofiles, provided that the heavy chain-light chain pairings of themolecule include the first and second selective recognition sites boundby the selective recognition agents. The broad applicability ofselective recognition agents that bind particular selective recognitionsites represents an improvement on methods that rely on anti-idiotypicagents that recognise differences at the level of the antigen bindingsite.

The methods of the present invention also represent an improvement overprior art methods that focus solely on addressing the problem ofmispairing between heavy chains of bispecific antibodies. The methodsdescribed herein allow multispecific antibodies having the correct heavychain-light chain pairings to be isolated, for example from a mixturecontaining antibodies having various combinations of pairings of heavyand light chains. The isolation of multispecific antibodies having onlythe correct pairings is achieved by virtue of the fact that eachselective recognition site includes residues derived from the heavychain and the light chain of the pairing. In the absence of the correctheavy chain-light chain pairings, the first and/or second selectiverecognition sites are not present such that these molecules are notrecognised and bound by the selective recognition agents. Onlymultispecific antibodies having the correct heavy chain-light chainpairings and thus presenting both selective recognition sites areselectively bound and captured by both first and second selectiverecognition agents in the first and second stages of the isolationmethod described herein.

A further advantage conferred by the methods of the present invention isthat the methods can exploit the natural variation found in the constantregions of heavy chains and light chains of different classes, subtypesand allotypes. As described above, the first and second heavy chains maybe different allotypes and therefore be distinguished by virtue of thenatural amino acid differences found in the constant regions, and inparticular the CH1 domains, of the naturally occurring heavy chainpolypeptides. Alternatively, or in addition, the first and second lightchains may belong to different classes e.g. the lambda and kappa lightchain classes. The differences in amino acid sequence between these twoclasses of light chain may be exploited in the production of selectiverecognition agents that selectively recognise either the first pairingor second pairing of the antibody molecule.

In view of the natural variation to be found in the constant regions ofdifferent heavy and light polypeptide chains, the need for extensiveprotein engineering of multispecific antibodies is minimised using themethods of the present invention. Furthermore, the fact that themultispecific antibodies can include naturally occurring heavy and lightpolypeptide chains or fragments thereof means that any unfavourableproperties associated with mutated immunoglobulin sequences e.g.unwanted immunogenicity can be avoided, and favourable properties, suchas the retention of natural effector function, can be preserved.

E. Reagents and Kits

The present invention also relates to reagents for producingmultispecific antibodies according to the present invention, andreagents, particularly antibodies, for isolating or purifyingmultispecific antibodies according to the present invention.Combinations of these reagents may be packaged in kit form.

Polynucleotides Encoding Fragments of Multispecific Antibodies

As described in section C above, the present invention includesexpression vectors comprising polynucleotide sequences encoding themultispecific antibodies provided herein. Such expression vectors may beincorporated into host cells or cell-free expression systems leading torecombinant expression of multispecific antibodies of the invention.

The present invention further includes polynucleotides or isolatedpolynucleotides encoding each of the separate immunoglobulin chains ofthe multispecific antibodies described herein. In addition, theinvention includes polynucleotides or isolated polynucleotides encodingfragments of each of the separate immunoglobulin chains, for examplefragments of the first and/or second heavy and light chains, wherein afragment is shorter than the full length immunoglobulin polypeptidechain by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 200 amino acid residues.

In certain embodiments, polynucleotides of the present invention encodefragments of the respective first and/or second heavy and/or lightchains wherein the fragment consists only of the constant region of eachchain. Polynucleotides may also encode fragments of the respective firstand/or second heavy and/or light chains wherein the fragment excludesthe polynucleotide regions encoding the CDR sequences. Polynucleotidesof the invention must encode fragments that are large enough to includethe specific residues from both the first and second heavy and lightchains that contribute to the first and second selective recognitionsites. Alternatively, polynucleotides of the invention must encodefragments of naturally occurring heavy and light chains that are longenough to encode a length of amino acid residues that differ by at leastone residue when produced as part of the first heavy chain and/or secondheavy chain.

Polynucleotides encoding fragments as described above may be useful asreagents for producing multispecific antibodies of the presentinvention. The polynucleotides may be inserted into plasmids or cloningvectors which allow for the introduction of polynucleotide sequencesencoding the portion or portions of the multispecific antibody whichis/are absent from the fragment. Suitable plasmids or vectors would beknown to those skilled in the art.

For example, polynucleotides encoding fragments consisting only of theconstant region of the heavy chain or light chain may be incorporatedinto a plasmid or cloning vector such that separate polynucleotidesencoding the VH and/or VL domains of antibodies of interest can beinserted in-frame into the open reading frame of the polynucleotideencoding the immunoglobulin chain constant regions. It would also bewithin the capabilities of the skilled person to take a polynucleotideencoding a fragment of a multispecific antibody lacking the CDRsequences and introduce polynucleotides encoding CDR sequences from twoor more antibodies of interest. The polynucleotides encoding fragmentsserve as the starting material for the production of multispecificantibodies having particular first and second selective recognitionsites associated with heavy chain-light chain pairings comprisingantigen binding sites targeting any antigens of interest.

Antibodies that Bind Multispecific Antibodies

As described in section D, the selective recognition agents used in thecontext of the methods of the present invention may be antibodies orantigen binding fragments thereof. All embodiments of the inventiondescribed above in the context of the methods of the invention areequally applicable to antibodies or antigen binding fragments providedherein as reagents for isolating or purifying multispecific antibodiesaccording to the invention.

The present invention further provides an antibody or antigen bindingfragment thereof that binds to a multispecific antibody as describedelsewhere herein, wherein the antibody or antigen binding fragmentthereof binds to an epitope, wherein the epitope is:

(i) comprised within the first pairing (the first heavy chain pairedwith the first light chain) but not the second pairing (the second heavychain paired with the second light chain); or

(ii) comprised within the second pairing (the second heavy chain pairedwith the second light chain) but not the first pairing (the first heavychain paired with the first light chain) and wherein the epitope doesnot include residues from the antigen binding site.

The epitope to which the antibody or antigen binding fragment thereofbinds is distinct or unique in that it is comprised only within eitherthe first heavy chain-light chain pairing or the second heavychain-light chain pairing of the multispecific antibody. Thus, incertain embodiments wherein the multispecific antibody comprises firstand second selective recognition sites comprised within the first andsecond heavy chain-light chain pairings, respectively, the epitope towhich the antibody binds comprises or consists of either the firstselective recognition site or the second selective recognition site. Asmentioned above, wherein the selective recognition agents of theinvention are antibodies, the term “selective recognition site” may beused interchangeably with the term “epitope”.

For multispecific antibodies of the invention comprising first andsecond heavy chains wherein the heavy chains differ by at least oneamino acid residue in the constant region, preferably the CH1 domain,the epitope to which the antibody binds may comprise at least one ofsaid residues that differs between the constant regions of the heavychains. In certain embodiments, the epitope to which the antibody bindsmay comprise or consist of 2, 3, 4, 5, 6 or 7 amino acids that differbetween the constant regions, preferably the CH1 domains, of the firstand second heavy chains.

Multispecific antibodies comprising first and/or second heavy chainswith fragments and/or amino acid substitutions derived from variousnaturally occurring heavy chain subtypes and allotypes are describedelsewhere herein. In certain embodiments, antibodies are provided thatbind to an epitope comprised either within the first heavy chain-lightchain pairing or the second heavy chain-light chain pairing of themultispecific antibody, wherein the epitope comprises one or moreresidues in the first or second heavy chain that differs between thesubtype and/or allotypes of the first and second heavy chains.

In certain embodiments, provided herein are antibodies or antigenbinding fragments that bind to a multispecific antibody of theinvention, wherein the epitope:

-   -   (i) is comprised within the first pairing and comprises one or        more amino acid residues selected from the following residues in        the first heavy chain: C131, R133, E137, S138, S178, N192 and/or        F193; or    -   (ii) is comprised within the second pairing and comprises one or        more amino acid residues selected from the following residues in        the second heavy chain: S131, K133, G137, G138, Y178, S192        and/or L193.

In certain embodiments, the epitope comprises a distinct combination ofamino acid residues in the heavy chain. For example, the epitope may becomprised within the first pairing and comprise the first heavy chainamino acid residues E137 and S138; or

-   -   (ii) comprised within the second pairing and comprise the second        heavy chain amino acid residues G137 and G138; or    -   (iii) comprised within the first pairing and comprise the first        heavy chain amino acid residues N192 and F193; or    -   (iv) comprised within the second pairing and comprises the        second heavy chain amino acid residues S192 and L193; or    -   (v) comprised within the first pairing and comprise the first        heavy chain amino acid residues R133, E137, S138, S178, N192 and        F193; or    -   (vi) comprised within the second pairing and comprise the second        heavy chain amino acid residues K133, G137, G138, S192, L193,        Y178.

The antibodies and antigen binding fragments thereof for use in theisolation or purification of multispecific antibodies according to thepresent invention may take any of the forms described above in thecontext of “selective recognition agents” for use in the methods of thepresent invention. For example, the antibodies may be camelid-derivedVHH antibodies. Alternatively, the antibodies may be conventionalantibodies, preferably conventional antibodies comprising at least onecomplementarity determining region in the VH domain or VL domain derivedfrom an antibody of a species in the family Camelidae. In particularlypreferred embodiments, the entire VH and/or VL domain of theconventional antibody is derived from a camelid species, preferably allama (Lama glama).

The present invention also provides polynucleotides encoding theantibodies and antigen binding fragments described herein, andexpression vectors comprising such polynucleotides operably linked toregulatory sequences which permit expression of the antibodies in a hostcell or cell-free expression system. Further provided are host cells orcell-free expression systems containing these expression vectors.

Kits

Also provided herein are kits with reagents for isolating multispecificantibodies according to the methods of the present invention. Inparticular, the present invention provides kits comprising first andsecond selective recognition agents, preferably wherein the first andsecond selective recognition agents are antibodies as describedelsewhere herein. First and second selective recognition agentsrecognising particular combinations of first and second selectiverecognition sites may be packaged together to be used for the isolationand/or purification of multispecific antibodies having specificcombinations of first and second selective recognition sites.

The kits comprising first and second selective recognition agents mayfurther include polynucleotide sequences (or plasmids or vectorscomprising the same) as described above wherein the polynucleotidesencode fragments of multispecific antibodies of the present invention,particularly fragments encoding at least the regions of themultispecific antibody incorporating first and second selectiverecognition sites. In this way, the kit can be used by one of skill inthe art to produce multispecific antibodies having distinct first andsecond selective recognition sites recognised by the first and secondselective recognition agents provided within the same kit. It followsthat kits of the invention may be used to produce multispecificantibodies as described herein, and also to isolate such multispecificantibodies according to the methods of the invention.

The kits may optionally include labelling and or instructional materialsproviding directions for practising the methods of the invention.

The invention will be further understood with reference to the followingnon-limiting examples.

EXAMPLES Example 1: Identification of Relevant Residues in CH1 Domains

The aim of this experiment was to identify heavy chain-only antibodies(VHHs) with the ability to specifically recognise and differentiatebetween distinct CH1-CL domain pairings. In particular, the aim was toidentify VHH antibodies having binding specificity for either a CH1-CLcomplex formed between a first heavy chain CH1 domain and the CL domainof a lambda light chain (referred to herein as CLambda) or a secondCH1-CL complex formed between a second heavy chain CH1 domain and akappa light chain (referred to herein as Ckappa). In order to achievethe desired binding specificity, the aim was to identify an epitopeformed by each pairing having residues contributed from both the CH1 andCLambda/Ckappa domains, and with at least one amino acid differencebetween the residues contributed from the first and second heavy chainCH1 domains.

The amino acid sequences of all publicly accessible CH1 domains fromIgG1, IgG2, IgG3 and IgG4 were aligned and variations in the amino acidsequences were identified. The amino acid variations or differencesbetween the different subtypes and allotypes were identified by doing asimple protein alignment (see FIG. 3 ). The location of these amino acidvariations in the three-dimensional structure of an antibody wasdetermined by mapping these positions onto solved Fab structures (Lambdaand Kappa Fabs).

Amino acid variation located at the surface of the CH1 domains andlocated in range of the Clambda and Ckappa chains was identified.Particular residues of interest (and combinations of residues)identified as suitable for contributing to epitopes distinguishingCH1/Clamdba and CH1/Ckappa pairings are listed below:

-   -   At position 131: residue C in the first heavy chain domain, and        residue S in the second heavy chain domain;    -   At position 133: residue R in the first heavy chain domain, and        residue K in the second heavy chain domain;    -   At position 137: residue E in the first heavy chain domain, and        residue G in the second heavy chain domain;    -   At position 138: residue S in the first heavy chain domain, and        residue G in the second heavy chain domain;    -   At positions 137 and 138: residues E and S in the first heavy        chain domain, and residues G and G in the second heavy chain        domain;    -   At position 178: residue S in the first heavy chain domain, and        residue Y in the second heavy chain domain;    -   At position 192: residue N in the first heavy chain domain and        residue S in the second heavy chain domain;    -   At position 193: residue F in the first heavy chain domain and        residue L in the second heavy chain domain;    -   At positions 192 and 193: residues N and F in the first heavy        chain domain, and residues S and L in the second heavy chain        domain.

Example 2: Generation of IgG with Specific Variation in the CH1 Pairedwith Clambda or Ckappa

Functional human IgG1 antibodies (binding to a target protein) havingeither a Lambda light chain (Antibody A, C, D and E) or a Kappa lightchain (antibody B and F) were mutated in their CH1 domain by mutagenesisto create the antibodies as indicated in Table 11:

TABLE 11 Mab A Mab B MabC Mab D Mab E Mab F (Lambda) (Kappa) (Lambda)(Lambda) (Lambda) (Kappa) R133K mutA1/ mutB1/ mutC1/ mutD1/ mutE1/mutF1/ Amut1 Bmut1 Cmut1 Dmut1 Emut1 Fmut1 E137G, mutA2/ mutB2/ mutC2/mutD2/ mutE2/ mutF2/ S138G Amut2 Bmut2 Cmut2 Dmut2 Emut2 Fmut2 N192S,mutA3/ mutB3/ mutC3/ mutD3/ mutE3/ mutF3/ F193L Amut3 Bmut3 Cmut3 Dmut3Emut3 Fmut3 R133K, mutA4/ mutB4/ mutC4/ mutD4/ mutE4/ mutF4/ E137G,Amut4 Bmut4 Cmut4 Dmut4 Emut4 Fmut4 S138G, N192S, F193L, S178Y S178YmutA5/ mutB5/ mutC5/ mutD5/ mutE5/ mutF5/ Amut5 Bmut5 Cmut5 Dmut5 Emut5Fmut5

The antibodies were produced separately by transient transfection ofmammalian expression vectors encoding the heavy chains and mammalianexpression vectors encoding the light chains into HEK cells. Six daysafter the transfection, the supernatant of the cells was collected, andthe antibody present was purified from the medium using standard ProteinA purification methods.

All the antibodies were produced without significant loss in yield ascompared to the non-mutagenised antibodies, suggesting no effect of themutations on the stability or capacity to bind protein A.

Example 3: Identification of VHH Antibodies that Recognise the IgGVariation in the CH1 Paired with Clambda or Ckappa

To identify VHH antibodies capable of recognising specific variation ina heavy chain CH1 domain paired with either a lambda or kappa CL domain,llama were immunized with the antibodies mutE1/Emut1, mutE2/Emut2,mutE3/Emut3 and mutE4/Emut4 (separately or in a mixture) or wereimmunized with mutF1/Fmut1, mutF2/Fmut2, mutF3/Fmut3 and mutF4/Fmut4(separately or in a mixture). To further increase the immune responsetowards the human CH1 domains containing the mutations, the human CH1,Clambda and Ckappa domains were fused to llama variable domains (VH,Vlambda and Vkappa) as well as llama Fc. Immunization was done by 6consecutive injections of antibodies mixed with non-complete Freundadjuvant, starting with two injections of 100 μg followed by fourinjections of 50 μg of antibody mixture.

After immunization, 400 ml of llama PBLs were isolated using standardficoll separation and the RNA was extracted using an RNA Qiagen kit.cDNA was synthesised using random hexamer primers and the superscriptIII transcriptase kit from LifeTechnologies. Genes encoding the VHHantibodies were cloned by PCR (using a technique similar to thatdescribed in the literature, in particular Roovers et al. Cancer ImmunolImmunother. 2007 March; 56(3):303-317) and cloned into phagemid vectorsto allow phage display selection.

After library construction, the phage expressing VHH antibodies able torecognise the mutation(s) in the CH1 domains in a lambda antibody wereselected by phage display using antibodies selected from: mutC1/Cmut1,mutC2/Cmut2, mutC3/Cmut3 or mutC4/Cmut4 (coated or biotinylated). Allthe selections were done in the presence of wild-type antibody (with nomutations in CH1) to avoid the identification of VHH antibodies lackingspecificity for the mutation(s) in CH1. In addition, in some casesselection was also carried out in the presence of an antibody containingthe same CH1 domain mutation but paired with a kappa light chain (calledcounter selection; for example using mutF4/Fmut4 when selection wasbeing carried out for VHH antibodies specific for mutC4/Cmut4). Thepurpose of this additional selection was to isolate VHH antibodieshaving an epitope specific to the mutated CH1-Clambda pairing.

The phage expressing VHH antibodies able to recognise the CH1mutation(s) in a Kappa antibody were selected by phage display usingantibodies selected from: mutF1/Fmut1, mutF2/Fmut2, mutF3/Fmut3 ormutF4/Fmut4 (coated or biotinylated). All the selections were done inthe presence of wild-type antibody (with no mutations in CH1) to avoidthe identification of VHH antibodies lacking specificity for themutation(s) in CH1. In addition, in some cases selection was alsocarried out in the presence of an antibody containing the same CH1domain mutation but paired with a lambda light chain (calledcounter-selection; for example using mutC4/Cmut4 when selection wasbeing carried out for VHH antibodies specific for mutF4/Fmut4). Thepurpose of this additional selection was to isolate VHH antibodieshaving an epitope specific to the mutated CH1-Ckappa pairing.

An example of the phage display selection rounds are indicated in Table12 below.

TABLE 12 ROUND 1 ROUND 2 PANNING COUNTER- IN-SOLUTION COUNTER- LIBRARIESSELECTION SELECTION SELECTION SELECTION 2x E-IIamas Cmut1 Cwt (+Fmut1)bio-Cmut1 Cwt (+Fmut1) Cmut2 Cwt (+Fmut2) bio-Cmut2 Cwt (+Fmut2) Cmut3Cwt(+Fmut3) bio-Cmut3 Cwt(+Fmut3) Cmut4 Cwt(+Fmut4) bio-Cmut4Cwt(+Fmut4) 2x F-IIamas Fmut1 Fwt (+Cmut1) bio-Fmut1 Fwt (+Cmut1) Fmut2Fwt (+Cmut2) bio-Fmut2 Fwt (+Cmut2) Fmut3 Fwt (+Cmut3) bio-Fmut3 Fwt(+Cmut3) Fmut4 Fwt (+Cmut4) bio-Fmut4 Fwt (+Cmut4)

Phage display selections were carried out using the antibodies shown inthe table above coated at 1 or 10 μg/ml on Maxisorp plates. Thecounter-selections were carried out with the antibodies indicated byadding an excess of the antibody in solution with the phage. Afterselection, the bound phage was removed by trypsin elution, and thenumber of phage were estimated by serially diluting the eluate in PBSand infecting TG1 bacteria. After bacterial infection, aliquots of TG1were spotted on agar plates containing ampicillin and glucose. Thenumber of colonies at each spot correlates with the number of phagepresent in the diluted aliquot. The results of the selection are shownin FIG. 10 . The phage dilutions are indicated above the images: 0,−1=10⁻¹, −2=10⁻², −3=10⁻³, −4=10⁴, −5=10⁻³.

Example 4: Screening of VHH Specific for the IgG Variation in the CH1Paired with Clambda or Ckappa

After selection (for example mutC4/Cmut4), the VHH antibodies of singleclones were produced and VHHs were tested (using ELISA) for binding to:

-   -   mutant antibodies having the correct light chain (for example        mutC4/Cmut4);    -   the same antibody without the mutation in the CH1 domain (for        example, a wild-type antibody);    -   the same antibody with a different mutation in the CH1 domain        (for example mutC1/Cmut1, mutC2/Cmut2 etc.); and    -   an antibody with a different light chain (for example        mutF4/Fmut4, mutF3/Fmut3 etc. . . . ).

For example VHH selected for mutC4/Cmut4 (lambda) were tested in ELISAfor binding to mutC4/Cmut4, wtC and mutF4/Fmut4. For example VHHselected for Fmut4 (kappa) were tested in ELISA for binding to Fmut4,wtF and Cmut4.

The ELISA results (OD) of some representative clones are indicated belowin Table 13.

TABLE 13 Coating for ELISA VHH clone Cmut4 Fmut4 Fwt Specificity 8G120.2 0.159 0.063 variant 4 => not OK 8G1 0.068 0.128 0.079 Variant 4 inpresence of Kappa light chain => OK 8G11 0.054 0.123 0.073 Variant 4 inpresence of Kappa light chain => OK 8B03 0.068 0.221 0.069 Variant 4 inpresence of Kappa light chain => OK 8GO9 0.068 0.153 0.114 F specific =>not OK

The sequences of the VHH clones in Table 13 are shown in Table 14.

TABLE 14 VHH SEQ Clone Sequence ID NO 8G12EVQLVESGGGLVRAGGSLRLSCAASGSIFGTTNMGW 15YRQAPGTQRDLVATINNGGITNYADSVKGRFTISTDYTKNTVYLQMDRLKPEDTAVYYCNAEYHFRPPSWGQ GTQVTVSS 8G1QVQLVESGGGLVQPGGSLRLSCRASGAIFSINHMGW 16YRQAPGKQRELVATITSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYSCNLDWTTGWGSRRD YWGQGTQVTVSS 8G11EVQLVESGGGLVQAGGSLRLSCAASGRTFDIYTMGW 17FRQVPGKEREFVADIGRAGGTTHYADSVKGRFAISRDNANDAVHLQMNSLKPEDTAVYYCATKVVPRAGRRL LDYDYWGQGTQVTVSS 8B03EVQLVESGGGLVQPGGSLRLSCRASGAIFSINHMGW 18YRQAPGKQRELVATITSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYSCNLDWTTGWGSRRD YWGQGTQVTVSS 8GO9QVQLVESGGGLVQAGGSLRLSCAASGRTFSRYGMSW 19FRQAPGKEREFVATISRGGSSTNYADSTKGRFTVSRDNAKNTVVLQMNSLKPEDTAVYYCAADTNWPVGAYE YWGQGTQVTVSS

In this example:

-   -   Clone 8G12 appears specific for the mut4 mutations (any of        R133K, E137G, S138G, N192S, F193L, S178Y);    -   Clones 8G1, 8G11 and 8603 are binding to Fmut4 (having a kappa        light chain) and not Cmut4 (having a lambda light chain) nor to        Fwt (with a kappa light chain) suggesting binding to the mut4        mutation in the context of a kappa light chain and thereby        indicating that their epitope is shared between the mutation and        the Kappa light chain;    -   Clone 8G09 binds to Fmut4 and also Fwt, suggesting that it binds        to the F antibody independently of the mutation.

To confirm that the VHH antibodies (i.e. the purification tools) arecapable of distinguishing between the CH1/Clambda pairing (or CH1/Ckappapairing) irrespective of the binding specificity of the antibody, thebinding of the VHH antibodies can be tested against several antibodieshaving different variable domains, and therefore differentfunctionalities, but the same mutation in the CH1 domains.

Example 5: Identification of VHH Antibodies that Recognise the IgGVariation in the CH1 Paired with Clambda or Ckappa

To identify more VHH antibodies with the desired specificity for CH1paired with Clambda or Ckappa, further llama immunizations were donewith only mut4 (Cmut4 or Fmut4). Immunization, RNA extraction andlibrary construction were carried out as described above.

Selection for clones from the phage libraries with specificity for theparticular CH1/CL pairs was carried out as indicated below in Table 15,with novel antibody preparations used for the selection. NotablyCmut4/Mix (which is the heavy chain of Cmut4 with a Kappa light chain ofthe F antibody) and Fmut4/mix (which is the heavy chain of Fmut4 with alambda light chain of the C antibody) were used also forcounter-selection in parallel to Cwt and Fwt counter-selection, whichallowed identification of mutation-specific VHH antibodies. These werescreened later to test if their epitope overlaps with the lambda orkappa light chain.

TABLE 15 R1 R2 R3 Immuni- Type Coating Counter- coating Counter- coatingCounter- zation sel R1 selection R2 selection R3 selection Cmut4 A1Cmut4 Cwt Cmut4 Cwt Fmut4/MIX Cwt (2 llamas) B1 Cmut4 Cmut4/Mix Cmut4Cmut4/Mix Fmut4/MIX Cwt Fmut4 A2 Fmut4 Fwt Fmut4 Fwt Cmut4/MIX Fwt (2llamas) B2 Fmut4 Fmut4/Mix Fmut4 Fmut4/Mix Cmut4/MIX Fwt

The results of the selection were assessed as described above in Example3 using phage titration and infection of TG1 bacterial cells, as shownin FIG. 11 . The phage titrations clearly show that specific VHHs areselected over non-coated wells (PBS) and that the presence of a counterselection agent (to remove unwanted VHH) has the desired effect.

Example 6: Screening of VHH Specific for the IgG Variation in the CH1Paired with Clambda or Ckappa

After selection, the VHH antibodies of single clones were produced andVHHs were screened by ELISA. The desired binding characteristics areindicated in Table 16

TABLE 16 Immuni- zation/ Selection Screening Cmut4 Coating: Cmut4 CwtFmut4 Fmut4/mix (lambda) (lambda) (Kappa) (lambda) Desired + − − +binding: Fmut4 Coating: Fmut4 Fwt Cmut4 Cmut4/mix (Kappa) (Kappa)(lambda) (Kappa) Desired + − − + binding:

Clones having the required binding characteristics in ELISA wereidentified from type selection A2 (6MP7 clones) and B2 (6MP8 clones)(see Table 17 below). Clones were scored positive when OD signal was at1.5 higher than the blank (no VHH added=BLANK).

TABLE 17 ELISA OD value on the indicated coated antibodies Fmut4 FwtCmut4/mix Cmut4 VHH clones (Kappa) (Kappa) (Kappa) (Lambda) 6MP8A8 0.1500.052 1.032 0.048 6MP8C10 0.122 0.048 0.813 0.046 6MP8C12 0.147 0.0510.927 0.049 6MP8C7 0.098 0.045 0.162 0.061 6MP8C8 0.434 0.046 0.1660.047 6MP8C9 0.732 0.045 0.172 0.047 6MP8D7 3.864 0.047 0.217 0.0486MP8D9 3.327 0.049 0.179 0.047 6MP8E9 0.387 0.047 0.205 0.047 BLANK 6MP80.051 0.046 0.054 0.048 6MP7E6 0.475 0.042 0.099 0.079 6MP7B2 0.4470.044 0.081 0.067 6MP7E10 0.114 0.044 0.079 0.065 6MP7E11 0.689 0.0510.077 0.05 BLANK 6MP7 0.051 0.066 0.046 0.061

Example 7: Purification of Antibodies Using the Purification Tools

VHH antibodies recognizing a specific epitope formed by the(variant-)CH1-CLambda pairing or formed by the (variant-)CH1-Ckappapairing were selected for the preparation of affinity purificationcolumns using conventional methods. For example, the VHH antibodies canbe coupled to sepharose beads and VHH-functionalized sepharose is packedinto small Tricorn columns, or a large 16K column with adaptors forsmall volumes. Analysis of the specificity of the columns is studiedusing chromatography and purified mAbs.

VHH-columns are equilibrated in PBS and the antibodies are injected onthe column in PBS. After washing off the unbound monoclonal antibodies(properly paired second arm of the bispecific and mispaired first armantibodies), bound monoclonal antibodies (containing the properly pairedfirst arm) are eluted with 50 mM glycine pH=2.0. The identity of theantibodies present in the elution can be confirmed functionally (inELISA or Biacore) or analytically by, for example, high resolution massspectrometry and SDS-Page.

The compatibility of the tools to purify bispecific antibodies (in thecase where one arm is a lambda and one arm is a kappa) is shown in Table18 below (L: Lambda; K: Kappa).

TABLE 18 Mut1 (K) Mut2 (K) Mut3 (K) Mut4 (K) wt (K) Mut1(L) — Y Y Y YMut2(L) Y — Y Y Y Mut3(L) Y Y — Y Y mut4(L) Y Y Y — Y wt (L) Y Y Y Y —

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims. Moreover, all embodiments described herein areconsidered to be broadly applicable and combinable with any and allother consistent embodiments, as appropriate.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

The invention claimed is:
 1. A composition comprising: (i) a firstpolynucleotide and a second polynucleotide encoding a first pairing of abispecific antibody, wherein the first polynucleotide encodes a heavychain comprising a first CH1 domain and the second polynucleotideencodes a light chain comprising a first CL domain, wherein the firstpairing comprises a first antigen binding site; and (ii) a thirdpolynucleotide and a fourth polynucleotide encoding a second pairing ofthe bispecific antibody, wherein the third polynucleotide encodes aheavy chain comprising a second CH1 domain and the fourth polynucleotideencodes a light chain comprising a second CL domain, wherein the secondpairing comprises a second antigen binding site, wherein: (a) the firstCH1 domain and the second CH1 domain are of different humanimmunoglobulin subtypes selected from the group consisting of IgG1,IgG2, IgG3 and IgG4, or the first CH1 domain and the second CH1domain—are of different human immunoglobulin allotypes selected from thegroup consisting of IGGH1.1 (SEQ ID NO: 1), IGGH1.3 (SEQ ID NO: 2),IGGH2 (SEQ ID NO: 3), IGGH2.2 (SEQ ID NO: 4), IGGH2.4 (SEQ ID NO: 5),IGGH4 (SEQ ID NO: 6), IGGH3 (SEQ ID NO: 7), IGG3.17 (SEQ ID NO: 8), andIGGH3.18 (SEQ ID NO: 9), (b) the first CL domain is a human lambda lightchain and the second CL domain is a human kappa light chain, wherein thehuman lambda light chain is of a human immunoglobulin allotype selectedfrom the group consisting of IGLC1*01 (SEQ ID NO: 10), IGLC2*01 (SEQ IDNO: 11), IGLC3*01 (SEQ ID NO: 12), IGLC7*01 (SEQ ID NO: 13), andIGLC6*01 (SEQ ID NO: 14), and (c) the first CH1 domain and the secondCH1 domain differ at one or more positions selected from the groupconsisting of positions 131, 133, 137, 138, 178, 192, and 193, accordingto the EU numbering scheme, wherein: (i) at position 131: C in the firstCH1 domain; S in the second CH1 domain; and/or (ii) at position 133: Rin the first CH1 domain; K in the second CH1 domain; and/or (iii) atposition 137: E in the first CH1 domain; Gin the second CH1 domain;and/or (iv) at position 138: S in the first CH1 domain; G in the secondCH1 domain; and/or (v) at position 178: S in the first CH1 domain; Y inthe second CH1 domain; and/or (vi) at position 192: N in the first CH1domain; S in the second CH1 domain; and/or (vii) at position 193: F inthe first CH1 domain; L in the second CH1 domain, in each case accordingto the EU numbering scheme.
 2. The composition of claim 1, wherein thefirst CH1 domain and the second CH1 domain of the bispecific antibodyare of different human immunoglobulin subtypes.
 3. The composition ofclaim 1, wherein the first CH1 domain and the second CH1 domain of thebispecific antibody are of different human immunoglobulin allotypes. 4.The composition of claim 1, wherein the antibody is monovalent.
 5. Thecomposition of claim 1, wherein each polynucleotide is comprised withinan expression vector.
 6. The composition of claim 5, wherein: (i) eachpolynucleotide is comprised within the same expression vector; (ii) eachpolynucleotide is comprised within a separate expression vector; (iii)the first polynucleotide and the second polynucleotide are comprisedwithin the same expression vector; (iv) the first polynucleotide and thethird polynucleotide are comprised within the same expression vector;(v) the first polynucleotide and the fourth polynucleotide are comprisedwithin the same expression vector; (vi) the second polynucleotide andthe third polynucleotide are comprised within the same expressionvector; (vii) the second polynucleotide and the fourth polynucleotideare comprised within the same expression vector; (viii) the thirdpolynucleotide and the fourth polynucleotide are comprised within thesame expression vector; (ix) the first polynucleotide, the secondpolynucleotide, and the fourth polynucleotide are comprised within thesame expression vector; (x) the first polynucleotide, the secondpolynucleotide, and the third polynucleotide are comprised within thesame expression vector; (xi) the first polynucleotide, the thirdpolynucleotide, and the fourth polynucleotide are comprised within thesame expression vector; or (xii) the second polynucleotide, the thirdpolynucleotide, and the fourth polynucleotide are comprised within thesame expression vector.
 7. A host cell comprising the composition ofclaim
 6. 8. A method of producing a bispecific antibody comprisingculturing the host cell of claim 7 so that the polynucleotides areexpressed and the bispecific antibody is produced.
 9. An isolatedpolynucleotide encoding a VHH antibody comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 15-18.
 10. A host cellcomprising the isolated polynucleotide of claim
 9. 11. A method ofproducing a VHH antibody comprising culturing the host cell of claim 10so that the polynucleotide is expressed and the VHH antibody isproduced.
 12. A host cell comprising: (i) a first polynucleotide and asecond polynucleotide encoding a first pairing of a bispecific antibody,wherein the first polynucleotide encodes a heavy chain comprising afirst CH1 domain and the second polynucleotide encodes a light chaincomprising a first CL domain, wherein the first pairing comprises afirst antigen binding site; and (ii) a third polynucleotide and a fourthpolynucleotide encoding a second pairing of the bispecific antibody,wherein the third polynucleotide encodes a heavy chain comprising asecond CH1 domain and the fourth polynucleotide encodes a light chaincomprising a second CL domain, wherein the second pairing comprises asecond antigen binding site, wherein: (a) the first CH1 domain and thesecond CH1 domain are of different human immunoglobulin subtypesselected from the group consisting of IgG1, IgG2, IgG3 and IgG4, or thefirst CH1 domain and the second CH1 domain—are of different humanimmunoglobulin allotypes selected from the group consisting of IGGH1.1(SEQ ID NO: 1), IGGH1.3 (SEQ ID NO: 2), IGGH2 (SEQ ID NO: 3), IGGH2.2(SEQ ID NO: 4), IGGH2.4 (SEQ ID NO: 5), IGGH4 (SEQ ID NO: 6), IGGH3 (SEQID NO: 7), IGG3.17 (SEQ ID NO: 8), and IGGH3.18 (SEQ ID NO: 9), (b) thefirst CL domain is a human lambda light chain and the second CL domainis a human kappa light chain, wherein the human lambda light chain is ofa human immunoglobulin allotype selected from the group consisting ofIGLC1*01 (SEQ ID NO: 10), IGLC2*01 (SEQ ID NO: 11), IGLC3*01 (SEQ ID NO:12), IGLC7*01 (SEQ ID NO: 13), and IGLC6*01 (SEQ ID NO: 14), and (c) thefirst CH1 domain and the second CH1 domain differ at one or morepositions selected from the group consisting of positions 131, 133, 137,138, 178, 192, and 193, according to the EU numbering scheme, wherein:(i) at position 131: C in the first CH1 domain; S in the second CH1domain; and/or (ii) at position 133: R in the first CH1 domain; K in thesecond CH1 domain; and/or (iii) at position 137: E in the first CH1domain; Gin the second CH1 domain; and/or (iv) at position 138: S in thefirst CH1 domain; G in the second CH1 domain; and/or (v) at position178: S in the first CH1 domain; Y in the second CH1 domain; and/or (vi)at position 192: N in the first CH1 domain; S in the second CH1 domain;and/or (vii) at position 193: F in the first CH1 domain; L in the secondCH1 domain, in each case according to the EU numbering scheme.
 13. Thehost cell of claim 12, wherein the first CH1 domain and the second CH1domain of the antibody are of different human immunoglobulin subtypes.14. The host cell of claim 12, wherein the first CH1 domain and thesecond CH1 domain of the antibody are of different human immunoglobulinallotypes.
 15. The host cell of claim 12, wherein the antibody ismonovalent.
 16. A method of producing a bispecific antibody comprisingculturing the host cell of claim 12 so that the polynucleotides areexpressed and the bispecific antibody is produced.